Expression of recombinant tetracycline efflux pumps for the production of lysine or lysine-derived products, and methods and applications thereof

ABSTRACT

The present invention provides a mutant polypeptide comprising the amino acid sequence of  Escherichia coli  tetracycline efflux pump A (TetA), and a polynucleotide encoding the polypeptide. The present invention provides a first expression plasmid vector comprising one or more first polynucleotides encoding a tetracycline efflux pump, one or more second polynucleotides independently selected from the group consisting of a third polynucleotides encoding a lysine decarboxylase polypeptide and a fourth polynucleotides encoding a lysine biosynthesis polypeptide, and a transformant comprising the expression plasmid vector. The present invention provides a host cell comprising one or more first, third or fourth polynucleotides as described herein integrated into a chromosome of the host cell. The present invention also provides a method for producing a lysine of a cadaverine using the transformant and/or the host cell.

BACKGROUND

Current approaches to improve lysine production and the production oflysine-derived products, such as cadaverine, focus on the overexpressionor attenuation of proteins involved in cellular metabolism. However, theyield obtained so far is not satisfying. Therefore, there is a need fornew techniques resulting in higher yields of lysine and cadaverine.

SUMMARY OF THE INVENTION

One aspect of the invention relates to a first polypeptide comprising,consisting of, or consisting essentially of a tetracycline efflux pumppolypeptide, a fragment thereof, or a mutant thereof. As used herein,the E. coli tetracycline efflux pump A is referred to as “TetA” and hasthe amino acid sequence of SEQ ID NO: 2. Examples of mutants of TetAinclude, without limitation, truncations of TetA such as TetA (aa1-185)having the polypeptide sequence of SEQ ID NO: 30 and TetA (aa1-96)having the polypeptide sequence of SEQ ID NO: 32. As used herein, “aa”refers to amino acid.

Another aspect of the invention relates to a non-naturally occurringfirst polynucleotide encoding one or more first polypeptides asdisclosed herein. As used herein, the E. coli tetracycline efflux pump Agene is referred to as “E. coli tetA” and comprises, consists of, orconsists essentially of the polynucleotide sequence of SEQ ID NO: 1.Examples of mutants of tetA include, without limitation, truncations oftetA such as tetA (nt 1-558) having the polynucleotide sequence of SEQID NO: 29 and tetA (nt 1-291) having the polynucleotide sequence of SEQID NO: 31. As used herein, “nt” refers to nucleotide.

Another aspect of the invention relates to a first expression plasmidvector comprising, consisting of, or consisting essentially of one ormore first polynucleotides as disclosed herein; and a backbone plasmidcapable of autonomous replication in a host cell, wherein the firstexpression plasmid vector is used for production of lysine or alysine-derived product. In certain embodiments, the first expressionplasmid vector further comprises one or more second polynucleotidesselected from the group consisting of a third polynucleotide encoding athird polypeptide comprising a lysine decarboxylase polypeptide, afragment thereof or a mutant thereof, and a fourth polynucleotideencoding a fourth polypeptide comprising a lysine biosynthesispolypeptide, a fragment thereof or a mutant thereof.

Another aspect of the invention relates to a transformant comprising,consisting of, or consisting essentially of one or more first expressionplasmid vectors as described herein in a host cell. In certainembodiments, the transformant as described herein, further comprises,consists of, or consists essentially of one or more second expressionplasmid vectors comprising, consisting of, or consisting essentially ofone or more fifth polynucleotides selected from the group consisting ofa first polynucleotide as disclosed herein, a third polynucleotide asdisclosed herein, and a fourth polynucleotide as disclosed herein; and abackbone plasmid capable of autonomous replication in a host cell,wherein the one or more second expression plasmid vectors are used forproduction of lysine or a lysine-derived product.

Another aspect of the invention relates to a mutant host cellcomprising, consisting of, or consisting essentially of one or morefirst polynucleotides as disclosed herein integrated into a chromosomeof a host cell. In certain embodiments, mutant host cell furthercomprises, consists of, or consists essentially of one or more secondpolynucleotides selected from the group consisting of thirdpolynucleotides as disclosed herein, and fourth polynucleotides asdisclosed herein.

Another aspect of the invention relates to a method for producing lysinecomprising obtaining a transformant and/or mutant host cell as disclosedherein, culturing the transformant and/or mutant host cell underconditions effective for the expression of the lysine; and harvestingthe lysine.

Another aspect of the invention relates to a method for producingcadaverine (1,5-pentanediamine) comprising cultivating a transformantand/or mutant host cell as disclosed herein, producing cadaverine usingthe culture obtained herein to decarboxylate lysine, and extracting andpurifying cadaverine using the culture obtained herein.

Other aspects of the invention relate to polyamides and1,5-diisocyanatopentane prepared from biobased cadaverine prepared asdisclosed herein, and compositions and preparation methods thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Polymerase chain reaction (PCR) primer sequences used toconstruct the recombinant expression plasmid vectors according to anembodiment of the invention. Primer sequences used to clone and truncatetetA, cadA, and various genes involved in the lysine biosyntheticpathway are provided.

FIG. 2: A table showing the plasmids and strains used in the Examples inaddition to the corresponding enzymes being overexpressed and the genesencoding the enzymes.

DETAILED DESCRIPTION OF THE INVENTION

The following description provides specific details for a thoroughunderstanding of, and enabling description for, embodiments of thedisclosure. However, one skilled in the art will understand that thedisclosure may be practiced without these details. In other instances,well-known structures and functions have not been shown or described indetail to avoid unnecessarily obscuring the description of theembodiments of the disclosure.

As disclosed herein, it has been found that expression of a tetracyclineefflux pump has resulted in unexpectedly high yields of lysine andcadaverine production. Furthermore, it has unexpectedly been found thatexpression of various mutants of the tetracycline efflux pump haveresulted in high cadaverine productions.

As used herein, the term “one or more” items (e.g. without limitation,polynucleotides, polypeptides, expression plasmid vectors, amino acids,nucleotides, mutations, plasmids, enzymes, proteins, sources, diamines,dicarboxylic acids, and polyamides) means that when there are aplurality of the items, said items may be the same or different.

One aspect of the invention relates to a first polypeptide comprising,consisting of, or consisting essentially of a tetracycline efflux pumppolypeptide, a fragment thereof, or a mutant thereof. In certainembodiments, the tetracycline efflux pump polypeptide comprises,consists of, or consists essentially of the amino acid sequence of SEQID NO: 2 (TetA), a fragment thereof, or a mutant thereof.

In certain embodiments, the first polypeptide comprises a tetracyclineefflux pump polypeptides selected from the group consisting of Tet,TetA, TetB, TetC, TetD, TetE, TetF, TetG, TetH, TetJ, TetK, TetL, TetM,TetO, TetP(A), TetP(B), TetQ, TetS, TetT, TetU, TetV, TetW, TetX, TetY,TetZ, TetA30, fragments thereof, and mutants thereof. For example,without limitation, the first polypeptide may comprise any of thetetracycline efflux pump polypeptides listed in Table 1, a fragmentthereof, or a mutant thereof.

TABLE 1 Genes that encode tetracycline efflux pumps and correspondinggenera Genes Polypeptides Source (Genus) tetA TetA Aeromonas,Citrobacter, Edwardsiella, Escherichia, Klebsiella, Plesimonas, Proteus,Pseudomonas, Salmonella, Serratia, Shigella, Vibrio tetB TetBActinobacillus, Aeromonas, Citrobacter, Enterobacter, Escherichia,Haempphilus. Klebsiella, Moraxella, Pasteurella, Plesimonas, Proteus,Providencia, Salmonella, Serratia, Shigella, Treponema, Vibrio, YersiniatetC TetC Citrobacter, Enterobacter, Escherichia, Proteus, Pseudomonas,Salmonella, Serratia, Shigella, Vibrio tetD TetC Aeromonas, Citrobacter,Edwardsiella, Enterobacter, Escherichia, Klebsiella, Pasteurella,Plesimonas, Salmonella, Shigella, Vibrio, Yersinia tetE TetE Aeromonas,Alcaligenes, Escherichia, Providencia, Pseudomonas, Serratia, Shigella,Vibrio tetF TetF Baceriodes fragilis tetG TetG Pseudomonas, Salmonella,Vibrio tetH TetH Pasteurella tetJ TetJ Proteus mirabilis tetK TetKBacillus, Clostridium, Enterococcus, Eubacterium, Haempphilus. Listeria,Mycobacterium, Nocardia, Peptostreptococcus, Staphylococcus,Streptococcus, Streptomyces tetL TetL Actinomyces, Bacillus,Clostridium, Enterococcus, Listeria, Mycobacterium, Peptostreptococcus,Staphylococcus, Streptococcus, Streptomyces tetM TetM Aerococcus,Actinomyces, Bacterionema, Bifidobacterium, Clostridium,Corynebacterium, Enterococcus, Eubacterium, Gardnerella, Gemella,Listeria, Mycoplasma, Peptostreptococcus, Staphylococcus, Streptococcus,Ureaplasma, Campylobacter, Eikenella, Fusobacterium, Haemophilus,Kingella, Neisseria, Pasteurella, Prevotella, Veillonella tetO TetOAerococcus, Enterococcus, Lactobacillus, Mobiluncus, Peptostreptococcus,Staphylococcus, Streptococcus, Campylobacter tetP(A) TetP(A)Clostridium, Helicobacter tetP(B) TetP(B) Clostridium tetQ TetQBaceriodes, Capnocytophaga, Mitsuokella, Porphyromonas, Prevotella,Veillonella, Eubacterium, Lactobacillus, Mobiluncus, Peptostreptococcus,Streptococcus tetS TetS Enterococcus, Lactobacillus, Listeria tetT TetTStreptococcus tetU TetU Enterococcus tetV TetV Mycobacterium tetW TetWButyrivibrio tetX TetX Baceriodes tetY TetY pIE1120 tetZ TetZCorynebacterium tetA(30) TetA(30) Agrobacterium

In certain embodiments, the first polypeptide comprises, consists of, orconsists essentially of the amino acid sequence of SEQ ID NO: 2 (TetA),a fragment thereof, or a mutant thereof. A mutant of TetA may include adeletion, substitution, addition, and/or insertion of one or more aminoacids to the amino acid sequence of SEQ ID NO: 2, while the mutant ofTetA provides substantially the same function as TetA (i.e., the mutantof TetA has about 80% or higher tetracycline efflux pump activitycompared to that of TetA; about 90% or higher tetracycline efflux pumpactivity compared to that of TetA; about 95% or higher tetracyclineefflux pump activity compared to that of TetA; about 97% or highertetracycline efflux pump activity compared to that of TetA; about 99% orhigher tetracycline efflux pump activity compared to that of TetA; orabout 100% or higher tetracycline efflux pump activity compared to thatof TetA.)

Examples of mutants of TetA include, without limitation, SEQ ID NO: 30(TetA (aa1-185; where “aa” refers to amino acids)) and SEQ ID NO: 32(TetA (aa1-96)). Other examples of TetA mutants may include TetA mutantsthat are truncated at structural loop regions connecting alpha heliceswithin the TetA polypeptide. In certain embodiments, TetA mutants mayinclude any truncations made in loop three of the TetA protein such asTetA aa1-97, TetA aa 1-98, TetA aa 1-99, TetA aa 1-100, TetA aa 1-101,TetA aa 1-102, TetA aa 1-103, or TetA aa 1-104. In certain embodiments,TetA mutants may include any truncations made in loop four of the TetAprotein such as TetA aa1-124, TetA aa 1-125, TetA aa 1-126, TetA aa1-127, TetA aa 1-128, TetA aa 1-129, TetA aa 1-130, TetA aa 1-131, TetAaa 1-132, or TetA aa 1-133. In certain embodiments, TetA mutants mayinclude any truncations made in loop five of the TetA protein such asTetA aa1-155, TetA aa 1-156, TetA aa 1-157, TetA aa 1-158, TetA aa1-159, TetA aa 1-160, TetA aa 1-161, or TetA aa 1-162. In certainembodiments, TetA mutants may include any truncations made in loop sixof the TetA protein such as TetA aa1-182, TetA aa 1-183, TetA aa 1-184,TetA aa 1-185, TetA aa 1-186, TetA aa 1-187, TetA aa 1-188, TetA aa1-189, TetA aa 1-190, TetA aa1-191, TetA aa 1-192, TetA aa 1-193, TetAaa 1-194, TetA aa 1-195, TetA aa 1-196, TetA aa 1-197, TetA aa 1-198,TetA aa 1-199, TetA aa1-200, TetA aa 1-201, TetA aa 1-202, TetA aa1-203, TetA aa 1-204, TetA aa 1-205, TetA aa 1-206, TetA aa 1-207, TetAaa 1-208, TetA aa 1-209, TetA aa 1-210, TetA aa 1-211, TetA aa 1-212,TetA aa 1-213, or TetA aa 1-214. In certain embodiments, TetA mutantsmay include any truncations made in loop seven of the TetA protein suchas TetA aa1-237, TetA aa 1-238, TetA aa 1-239, TetA aa 1-240, TetA aa1-241, TetA aa 1-242, TetA aa 1-243, TetA aa 1-244, or TetA aa 1-245. Incertain embodiments, TetA mutants may include any truncations made inloop eight of the TetA protein such as TetA aa1-268, TetA aa 1-269, TetAaa 1-270, TetA aa 1-271, TetA aa 1-272, TetA aa 1-273, TetA aa 1-274,TetA aa 1-275, TetA aa 1-276, TetA aa 1-277, or TetA aa 1-278. Incertain embodiments, TetA mutants may include any truncations made inloop nine of the TetA protein such as TetA aa1-321, TetA aa 1-322, TetAaa 1-323, TetA aa 1-324, TetA aa 1-325, TetA aa 1-326, TetA aa 1-327,TetA aa 1-328, TetA aa 1-329, TetA aa 1-330, TetA aa 1-331, TetA aa1-332, TetA aa 1-333, TetA aa 1-334, TetA aa 1-335, TetA aa 1-336, TetAaa 1-337, TetA aa 1-338, or TetA aa 1-339. In certain embodiments, TetAmutants may include any truncations made in loop ten of the TetA proteinsuch as TetA aa1-360, TetA aa 1-361, TetA aa 1-362, TetA aa 1-363, TetAaa 1-364, TetA aa 1-365, TetA aa 1-366, or TetA aa 1-367.

As used herein, a polypeptide comprising a specific polypeptide sequencemay include fragments, and/or mutants of the specific polypeptidesequence, while still providing substantially the same function as thewhole original unmutated specific polypeptide sequence. A fragment of apolypeptide means a part of the polypeptide that provides substantiallythe same function as the whole polypeptide. Examples of mutants of aspecific polypeptide sequence include deletions, substitutions,additions, and/or insertions of one or more amino acids to the specificpolypeptide sequence. For example, a fragment or mutant of TetApossesses substantially the same function of the TetA polypeptide (e.g.tetracycline efflux pump activity).

Another aspect of the invention relates to a first polynucleotideencoding one or more first polypeptides that are the same or differentas disclosed herein. In one embodiment, the first polypeptide comprises,consists of, or consists essentially of a tetracycline efflux pumppolypeptide, a fragment thereof or a mutant thereof. When there are aplurality of the first polypeptides, each first polypeptide may be thesame or different, and may be expressed individually or as a fusionprotein.

In certain embodiments, the first polynucleotide sequence preferablycomprises one or more of a E. coli tetracycline efflux pump gene tetA(SEQ ID NO: 1), a fragment thereof, and/or a mutant thereof. In certainembodiments, the first polynucleotide may encode any of the tetracyclineefflux pumps as disclosed herein. In certain embodiments, the firstpolynucleotide sequence may be selected from the group consisting of SEQID NO: 29 (tetA (nt 1-558)), SEQ ID NO: 31 (tetA (nt 1-291), and codonoptimized tetA's.

In certain embodiments, the first polynucleotide sequence comprises one,two, three, four, five, six, seven, eight, nine, or ten tetracyclineefflux pump genes independently selected from the group consisting oftet, tetA, tetB, tetC, tetD, tetE, tetF, tetG, tetH, tetJ, tetK, tetL,tetM, tetO, tetP(A), tetP(B), tetQ, tetS, tetT, tetU, tetV, tetW, tetX,tetY, tetZ, tetA30, fragments thereof, and mutants thereof. For example,the first polynucleotide sequence may, without limitation, comprise anyof the tetracycline efflux pump genes listed in Table 1, a fragmentthereof, or a mutant thereof. In certain embodiments, the tetracyclineefflux pump genes may, without limitation, be from any of thecorresponding genera listed in Table 1.

In certain embodiments, the first polynucleotide comprises, consists of,or consists essentially of the polynucleotide tetracycline efflux pumpgene tetA (SEQ ID NO: 1), a mutant thereof, or a fragment thereof. Amutant of tetA may include a deletion, a substitution, an addition,and/or an insertion of one or more nucleotides to the polynucleotidesequence of SEQ ID NO: 1, while the protein encoded by the mutant oftetA provides substantially the same function as TetA (i.e., the mutantof TetA has about 80% or higher tetracycline efflux pump activitycompared to that of TetA; about 90% or higher tetracycline efflux pumpactivity compared to that of TetA; about 95% or higher tetracyclineefflux pump activity compared to that of TetA; about 97% or highertetracycline efflux pump activity compared to that of TetA; about 99% orhigher tetracycline efflux pump activity compared to that of TetA; orabout 100% or higher tetracycline efflux pump activity compared to thatof TetA).

In certain embodiments, the first polynucleotide may be a recombinant ornon-naturally occurring polynucleotide. In certain embodiments, thefirst polynucleotide may be cDNA. In certain embodiments, the firstpolynucleotide may be obtained by codon optimization for optimalpolypeptide expression in a particular microorganism (e.g., E. coli, H.alvei, or P. aeruginosa).

Nucleotide sequences, polynucleotides, and DNA molecules as used hereinare not limited to the functional region, and may include at least oneof an expression suppression region, a coding region, a leader sequence,an exon, an intron, and an expression cassette (see, e.g. Papadakis etal., “Promoters and Control Elements: Designing Expression Cassettes forGene Therapy,” Current Gene Therapy (2004), 4, 89-113). Furthermore,nucleotide sequences or polynucleotides may include double strand DNA orsingle strand DNA (La, a sense chain and an antisense chain constitutingthe double strand DNA), or RNA. A polynucleotide containing a specificpolynucleotides sequence may include fragments, and/or mutants of thespecific polynucleotides sequence. A fragment of a polynucleotide meansa part of the polynucleotide that encodes a polypeptide which providessubstantially the same function as the polypeptide encoded by the wholepolynucleotide. Examples of mutants of a specific polynucleotidessequence include naturally occurring allelic mutants; artificialmutants; and polynucleotides sequences obtained by deletion,substitution, addition, and/or insertion of one or more nucleotides tothe specific polynucleotides sequence. It should be understood that suchfragments, and/or mutants of a specific polynucleotides sequence encodepolypeptides having substantially the same function as the polypeptideencoded by the original, specific polynucleotides sequence. For example,a fragment and/or mutant of tetA encodes a polypeptide that possessessubstantially the same function of TetA (e.g. tetracycline efflux pumpactivity).

Codon optimization is a technique that may be used to maximize theprotein expression in an organism by increasing the translationalefficiency of the gene of interest. Different organisms often showparticular preferences for one of the several codons that encode thesame amino acid due to mutational biases and natural selection. Forexample, in fast growing microorganisms such as E. coli, optimal codonsreflect the composition of their respective genomic tRNA pool.Therefore, the codons of low frequency of an amino acid may be replacedwith codons for the same amino acid but of high frequency in the fastgrowing microorganism. Accordingly, the expression of the optimized DNAsequence is improved in the fast growing microorganism. See, e.g.http://www.guptalab.org/shubhg/pdf/shubhra_codon.pdf for an overview ofcodon optimization technology, which is incorporated herein by referencein its entirety. As provided herein, polynucleotide sequences may becodon optimized for optimal polypeptide expression in a particularmicroorganism including, but not limited to, E. coli, H. alvei, and P.aeruginosa.

In certain embodiments, mutants of a polynucleotide can be obtained fromcodon optimization of the polynucleotide to decrease the guanine (G) andcytosine (C) polynucleotide content thereof for improved proteinexpression. A genome is considered GC-rich if about 50% or more of itsbases are G or C. A high GC content in the polynucleotide sequence ofinterest may lead to the formation of secondary structure in the mRNA,which can result in interrupted translation and lower levels ofexpression. Thus, changing G and C residues in the coding sequence to Aand T residues without changing the amino acids may provide higherexpression levels.

Another aspect of the invention relates to a first expression plasmidvector comprising, consisting of, or consisting essentially of:

one or more first polynucleotides that are the same or different, andeach encodes one or more first polypeptides that are the same ordifferent and each comprises, consists of, or consists essentially of atetracycline efflux pump polypeptide, a fragment thereof, or a mutantthereof, and

a backbone plasmid capable of autonomous replication in a host cell,

wherein the first expression plasmid vector is used for production oflysine or a lysine-derived product.

In one embodiment, there are a plurality of the first polypeptides, eachfirst polypeptide may be the same or different, and may be expressedindividually or as a fusion protein.

As used herein, the term “host cell” refers to a microorganism cell thatmay be any cell that can be transformed with an expression plasmidvector (e.g., Pseudomonas (e.g., P. aeruginosa), Escherichia (e.g., E.coli), Corynebacterium (e.g., Corynebacterium glutamicum), Bacilli,Hafnia (e.g., Hafnia alvei), Brevibacterium, Lactobacillus (e.g.,Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillussaerimneri), Lactococcus (e.g., Lactococcus lactis, Lactococcus lactisssp. cremoris, Lactococcus lactis ssp. lactis), and Streptococcus (e.g.,Streptococcus thermophilus)).

An E. coli cell may be any of the E. coli strains derived from E. coliK12 (e.g., MG1655, W3110, DH10b, DH1, BW2952 and strains derivedtherefrom) or E. coli B, or strains derived therefrom.

In certain embodiments, the host cell may contain one or more endogenousplasmids. In certain embodiments, the host cell does not containendogenous plasmids. The term “cure” as used herein means to remove oneor more endogenous plasmids from a host cell. In certain embodiments, ahost cell may be “cured” of all endogenous plasmids by removing allendogenous plasmids from the host cell. In certain embodiments, a hostcell may be “cured” of one or more endogenous plasmids by removing onlythe one or more endogenous plasmids that is targeted for removal fromthe cell.

In certain embodiments, the host cell may be a prokaryotic cell (e.g.is, H. alvei) containing endogenous plasmids that encode specifictoxin/antitoxin gene pairs. Such toxin/antitoxin gene pairs play a rolein maintenance of the genetic information and response to stress. (See,Wertz et al. “Chimeric nature of two plasmids of Hafnia alvei encodingthe bacteriocins alveicins A and B.” Journal of Bacteriology, (2004)186: 1598-1605.) As long as the cell has one or more plasmids comprisingan antitoxin gene, the toxin is neutralized by the antitoxin that iscontinuously expressed by the one or more plasmids to keep the cellsalive. In certain prokaryotes, the antitoxin protein degrades fasterthan the toxin protein. If the plasmid comprising the antitoxin gene islost from the cell, the toxin protein will exist longer than theantitoxin protein in the cell and kill or inhibit the growth of thecell. Therefore, plasmids comprising the antitoxin or thetoxin/antitoxin gene are preferably maintained to keep the host cellalive.

As used herein, a toxin/antitoxin gene pair has two genes, one is atoxin gene which expresses a polypeptide toxic to a host cell, and theother is an antitoxin gene which neutralizes the toxic polypeptide inthe host cell. Examples of the toxin/antitoxin gene pair include,without limitation, abt/abi gene pair and aat/aai gene pair, fragmentsthereof, and mutants thereof. In some embodiments, the toxinpolynucleotide sequence comprises, consists of, or consists essentiallyof the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 14, fragmentsthereof, or mutants thereof. In some embodiments, the antitoxinpolynucleotide sequence comprises, consists of, or consists essentiallyof the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 15, fragmentsthereof, or mutants thereof.

In certain embodiments, the host cell may be any H. alvei strain, e.g.,endogenous plasmid-free H. alvei strains or H. alvei strains containingendogenous plasmids. For example, the host cell may be an H. alveistrain containing one or more pAlvA plasmids or the cured strainsthereof (pAlvA-strains), or an H. alvei strain containing one or morepAlvB plasmids and the cured strains thereof (pAlvB-strains).

In certain embodiments, the expression plasmid vector disclosed herein(e.g. the first expression plasmid vector) may further comprise one ormore antitoxin genes independently selected from the group consisting ofabi gene, aai gene, mutations and fragments thereof, and/or one or moretoxin/antitoxin gene pairs independently selected from the groupconsisting of abt/abi gene pair and aat/aai gene pair, and mutations andfragments thereof. For example, in certain embodiments, an expressionplasmid vector (e.g. the first expression plasmid vector) may furthercomprise an antitoxin polynucleotide that counteracts a toxinpolypeptide that is harmful to the host cell, and a toxin polynucleotidesequence encoding the toxin polypeptide.

In certain embodiments, the host cell is an industrial strain suitableto be used in industrial-scale or large-scale production. For example,industrial strains may be cultivated in a fermenter. The scale ofculture may range from hundreds of liters to millions of liters. On theother hand, a laboratory strain usually is cultivated in a few liters orless. In certain embodiments, an industrial strain may grow in a simpleror more economical medium than laboratory strains.

A backbone plasmid capable of autonomous replication in a host cell maybe any plasmid that can replicate in the host cell. In one embodiment,an expression plasmid vector comprises a backbone plasmid that canreplicate in E. coli. In another embodiment, an expression plasmidvector comprises a backbone plasmid that can replicate in H. alvei.Examples of the backbone plasmids include, without limitation, backboneplasmids that can replicate in E. coli strains, e.g. pUC (e.g. pUC18 andpUC19 plasmids), pBR322, pSC101, p15a, pACYC, pET, and pSC101 plasmids,and plasmids derived therefrom.

In certain embodiments, the mutants of a polynucleotide can be obtainedfrom codon optimization of the polynucleotide for a particularmicroorganism (e.g., E. coli, H. alvei, or P. aeruginosa) to enhancepolypeptide expression.

In certain embodiments, the first expression plasmid vector may be usedfor the production of lysine or a lysine derived product as describedherein. In certain embodiments, a lysine derived product may becadaverine as described herein.

In certain embodiments, the first expression plasmid vector furthercomprises one or more sixth polynucleotides that are the same ordifferent, and each encodes a sixth polypeptide that comprises, consistsof, or consists essentially of an antibiotic resistance protein, afragment, or a mutant thereof. When there are a plurality of the sixthpolynucleotides, the sixth polypeptides encoded by the sixthpolynucleotides may be the same and different, and may be expressedindividually or as a fusion protein.

In certain embodiments, the antibiotic resistance protein may be atetracycline resistance protein or an oxytetracycline-resistanceprotein. In certain embodiments, the antibiotic resistance protein maybe selected from the group that comprises, consists of, or consistsessentially of OtrA, OtrB, OtrC, Tcr3, a fragment, and/or mutantthereof. In certain embodiments, OtrA may be from the genusMycobacterium or Streptomyces. In certain embodiments, OtrB, OtrC, andTcr3 may be from the genus Streptomyces. In certain embodiments, thesixth polynucleotide may be selected from the group that comprises,consists of, or consists essentially of otrA, otrB, otrC, tcr3, afragment, and/or mutant thereof. In certain embodiments, otrA may befrom the genus Mycobacterium or Streptomyces. In certain embodiments,otrB, otrC, and tcr3 may be from the genus Streptomyces.

In certain embodiments, the first expression plasmid vector comprising,consisting of, or consisting essentially of:

one or more first polynucleotides that are the same or different, andeach encodes one or more first polypeptides that are the same ordifferent and each comprises, consists of, or consists essentially of atetracycline efflux pump polypeptide, a fragment thereof, or a mutantthereof;

one or more second polynucleotides that are the same or different andindependently selected from the group consisting of:

-   -   a third polynucleotide encoding a third polypeptide comprising,        consisting of, or consisting essentially of a lysine        decarboxylase polypeptide, a fragment thereof or a mutant        thereof, and    -   a fourth polynucleotide encoding a fourth polypeptide        comprising, consisting of, or consisting essentially of a lysine        biosynthesis polypeptide, a fragment thereof or a mutant        thereof; and

a backbone plasmid capable of autonomous replication in a host cell,

-   -   wherein the first expression plasmid vector is used for        production of lysine or a lysine-derived product.

In certain embodiments, when there are a plurality of the secondpolynucleotides, each third polypeptide may be the same or different andeach fourth polypeptide may be the same or different; the third andfourth polypeptides may be expressed individually or as a fusionprotein.

In certain embodiments, the third polynucleotide encodes a thirdpolypeptide that comprises, consists of, or consists essentially of alysine decarboxylase polypeptide, a fragment thereof, or a mutantthereof.

In certain embodiments, the third polynucleotide may be selected fromthe group that comprises, consists of, or consists essentially of SEQ IDNO: 41 (cadA), SEQ ID NO: 42 (ldcC), SEQ ID NO: 8 (ldc2), fragmentsthereof, and mutants thereof. In certain embodiments, thirdpolynucleotide is selected from the group that comprises, consists of,or consists essentially of SEQ ID NO: 10 (ldc2 co-1), SEQ ID NO: 34(ldc2 co-1 C332G), SEQ ID NO: 35 (ldc2 co-1 A785C), SEQ ID NO: 36 (ldc2co-1 A795C), SEQ ID NO: 37 (ldc2 co-1 C332G/A785C), SEQ ID NO: 38 (ldc2co-1 C332G/A795C), SEQ ID NO: 39 (ldc2 co-1 A785C/A795C), and SEQ ID NO:40 (ldc2 co-1 C332G/A785C/A795C).

In certain embodiments, the third polypeptide may comprise, consist of,or consist essentially of Escherichia coli CadA (SEQ ID NO: 6),Escherichia coli LdcC (SEQ ID NO: 7), Pseudomonas aeruginosa Ldc2 (SEQID NO: 9), a fragment thereof, or a mutant thereof. In certainembodiments, the lysine decarboxylase may be a lysine decarboxylase froma species that is homologous to E. coli LdcC or CadA. For example, thelysine decarboxylase may be Shigella sonnei CadA or Salmonella entericalysine decarboxylase, a fragment, or a mutant thereof.

Examples of mutants of Ldc2 include, without limitation, SEQ ID NO: 11(Ldc2 S111C), SEQ ID NO: 16 (Ldc2 N262T), SEQ ID NO: 17 (Ldc2 K265N),SEQ ID NO: 18 (Ldc2 S111C/N262T), SEQ ID NO: 19 (Ldc2 S111C/K265N), SEQID NO: 20 (Ldc2 N262T/K265N), and SEQ ID NO: 21 (Ldc2S111C/N262T/K265N), homologous polypeptides of Ldc2, homologouspolypeptides of Ldc2 S111C (e.g. Ldc2 S111X), homologous polypeptides ofLdc2 N262T (e.g. Ldc2 N262X′), homologous polypeptides of Ldc2 K265N(e.g. Ldc2 K265X′), homologous polypeptides of Ldc2 S111C/N262T (e.g.Ldc2 S111X/N262X′), homologous polypeptides of Ldc2 S111C/K265N (e.g.Ldc2 S111X/K265X″), homologous polypeptides of Ldc2 N262T/K265N (e.g.Ldc2 N262X′/K265X″), and homologous polypeptides of Ldc2S111C/N262T/K265N (e.g. Ldc2 S111X/N262X′/K265X″). X is any amino acidthat is not serine, X′ is any amino acid that is not asparagine, and X″is any amino acid that is not lysine. As used herein, a homologouspolypeptide is at least about 90%, at least about 95%, at least about97%, at least about 98%, or at least about 99% homologous with thepolypeptide. When a Ldc2 mutant has multiple mutations, each mutationmay be the same or different.

In certain embodiments, the third polypeptides are mutants of Ldc2, andthe corresponding third polynucleotides encoding the third polypeptidesare polynucleotides encoding Ldc2 (e.g. ldc2 (SEQ ID NO: 8)), a codonoptimized ldc2 (e.g. ldc2-col, SEQ ID NO: 10)) containing one or moresuitable nucleotide mutations that are the same or different andindependently selected from the group consisting of a mutation atnucleotide position 331, a mutation at nucleotide position 332, amutation at nucleotide position 333, a mutation at nucleotide position784, a mutation at nucleotide position 785, a mutation at nucleotideposition 786, a mutation at nucleotide position 793, a mutation atnucleotide position 794, and a mutation at nucleotide position 795.

In certain embodiments, the third polypeptides are mutants of Ldc2, andthe corresponding third polynucleotides encoding the third polypeptidesare polynucleotides encoding Ldc2 (e.g. ldc2 (SEQ ID NO: 8), a codonoptimized ldc2 (e.g. ldc2-col, SEQ ID NO: 10)) containing one or moresuitable nucleotide mutations that are the same or different andindependently selected from the group consisting of a mutation atnucleotide position 332, a mutation at nucleotide position 785, and amutation at nucleotide position 795. In certain examples, withoutlimitation, the nucleotide at position 332 may be mutated to G, thenucleotide at position 785 may be mutated to a C, and the nucleotide atposition 795 may be mutated to a T or C.

In certain embodiments, a lysine decarboxylase polypeptide may include adeletion, substitution, addition, and/or insertion of one or more aminoacids to the amino acid sequence of a lysine decarboxylase polypeptide,while the mutant of lysine decarboxylase polypeptide providessubstantially the same function as a lysine decarboxylase polypeptide(i.e., the mutant of a lysine decarboxylase polypeptide has about 80% orhigher lysine decarboxylase activity compared to that of a lysinedecarboxylase polypeptide; about 90% or higher lysine decarboxylaseactivity compared to that of a lysine decarboxylase polypeptide; about95% or higher lysine decarboxylase activity compared to that of a lysinedecarboxylase polypeptide; about 97% or higher lysine decarboxylaseactivity compared to that of a lysine decarboxylase polypeptide; about99% or higher lysine decarboxylase activity compared to that of a lysinedecarboxylase polypeptide; or about 100% or higher lysine decarboxylaseactivity compared to that of a lysine decarboxylase polypeptide).

In certain embodiments, the fourth polynucleotide encodes a fourthpolypeptide that comprises, consists of, or consists essentially of alysine biosynthesis polypeptide, a fragment thereof, or a mutantthereof. In certain embodiments, the fourth polynucleotide may be a geneselected from the group consisting of sucA, ppc, aspC, lysC, asd, dapA,dapB, dapD, argD, dapE, dapF, lysA, ddh, pntAB, cyoABE, gadAB, ybjE,gdhA, gltA, sucC, gadC, acnB, pflB, thrA, aceA, aceB, gltB, aceE, sdhA,murE, speE, speG, puuA, puuP, ygjG, fragments thereof, and mutantsthereof. For example, without limitation, the fourth polynucleotide maycomprise the sequence of any one of the E. coli genes, fragmentsthereof, or mutants thereof, listed in Table 2.

In certain embodiments, the fourth polynucleotide may comprise thesequence of a gene involved in lysine biosynthesis that is homologous toany one of the genes listed in Table 2. For example, the fourthpolynucleotide may comprise the sequence of a gene involved in lysinebiosynthesis that is from a species other than E. coli. In certainembodiments, the fourth polynucleotide may comprise the sequence of apolynucleotide sequence decoding the aspartokinase, LysC, fromStreptomyces lividans (GenBank EOY48571.1). As used herein, a gene thatis homologous to an E. coli gene has a polynucleotide sequence with atleast about 90%, at least about 95%, at least about 97%, at least about98%, or at least about 99% sequence homology with the polynucleotidesequence of the E. coli gene.

TABLE 2 E. coli Proteins/genes involved in lysine biosynthesis. ProteinGene GenBank Accession No. α-ketogultarate dehydrogenase (SucA) sucAYP_489005.1 Phosphoenolpyruvate carboxylase (PPC) ppc AAC76938.1aspartate transaminase (AspC) aspC AAC74014.1 aspartate kinase (LysC)lysC NP_418448.1 aspartate semialdehyde dehydrogenase asd AAC76458.1(Asd) dihydrodipicolinate synthase (DapA) dapA NP_416973.1dihydropicolinate reductase (DapB) dapB AAC73142.1 tetrahydrodipicoinatesuccinylase (DapD) dapD AAC73277.1 N-succinyldiaminopimelate argDAAC76384.1 aminotransferase (ArgD) N-succinyl-L-diaminopimelatedeacylase dapE AAC75525.1 (DapE) diaminopimelate epimerase (DapF) dapFAAC76812.2 diaminopimelate decarboxylase (LysA) lysA AAC75877.1meso-diaminopimelate dehydrogenase ddh P04964.1 (Ddh) pyridinenucleotide transhydrogenase pntAB AAC74675.1, AAC74674.1 (PntAB)cytochrome O oxidase (CyoABE) cyoABE AAC73535.1, AAC73534.1, AAC73531.1glutamate decarboxylase (GadAB) gadAB AAC76542.1, AAC74566.1 L-aminoacid efflux transporter (YbjE) ybjE AAC73961.2 glutamate dehydrogenase(GdhA) gdhA AAC74831.1 citrate synthase (GltA) gltA AAC73814.1succinyl-coA synthase (SucC) sucC AAC73822.1 glutamate-GABA antiporter(GadC) gadC AAC74565.1 aconitase B (AcnB) acnB AAC73229.1pyruvate-formate lyase (PflB) pflB AAC73989.1 aspartatekinase/homoserine thrA AAC73113.1 dehydrogenase (ThrA) isocitrate lyase(AceA) aceA AAC76985.1 malate synthase (AceB) aceB AAC76984.1 glutmatesynthase (GltB) gltB AAC76244.2 pyruvate dehydrogenase (AceE) aceEAAC73225.1 succinate dehydrogenase (SdhA) sdhA AAC73817.1UDP-N-acetylmuramoyl-L-alanyl-D- murE AAC73196.1glutamate:meso-diaminopimelate ligase (MurE) putrescine/cadaverine speEAAC73232.1 aminopropyltransferase (SpeE) spermidine acetyltransferase(SpeG) speG AAC74656.1 glutamate-putrescine/glutamate- puuA AAC74379.2cadaverine ligase (PuuA) putrescine importer (PuuP) puuP AAC74378.2putrescine/cadaverine aminotransferase ygjG AAC76108.3 (YgjG)

In certain embodiments, the fourth polypeptide comprises, consists of,or consists essentially of a lysine biosynthesis polypeptide, a fragmentthereof or a mutant thereof. In certain embodiments, the fourthpolypeptide may be selected from the group consisting of SucA, Ppc,AspC, LysC, Asd, DapA, DapB, DapD, ArgD, DapE, DapF, LysA, Ddh, PntAB,CyoABE, GadAB, YbjE, GdhA, GltA, SucC, GadC, AcnB, PflB, ThrA, AceA,AceB, GltB, AceE, SdhA, MurE, SpeE, SpeG, PuuA, PuuP, and YgjG,fragments thereof, and mutants thereof. For example, without limitation,the fourth polypeptide may be any one of the proteins listed in Table 2.In certain embodiments, the fourth polypeptide may contain one or moremutations. For example, the fourth polypeptide may comprise the sequenceof the E. coli aspartokinase III (LysC or AKIII) polypeptide with amutation from a methionine to an isoleucine at position 318 and amutation from a glycine to an aspartic acid at position 323 (LysC-1(M318I, G323D)) having the sequence of SEQ ID NO: 26. In certainembodiments, the fourth polypeptide may comprise the sequence of the E.coli LysC polypeptide with a mutation from a threonine to a methionineat position 344 and a mutation from a threonine to an isoleucine atposition 352 (LysC-1 (T344M, T352I)) having the sequence of SEQ ID NO:27.

In certain embodiments, the fourth polypeptide may comprise the sequenceof a protein involved in lysine biosynthesis that is homologous to anyone of the proteins listed in Table 2. In certain embodiments, thefourth polynucleotide may comprise the sequence of a protein involved inlysine biosynthesis that is from a species other than E. coli. Forexample, the fourth polypeptide may comprise the sequence of theaspartokinase protein, LysC, from Streptomyces lividans (GenBankEOY48571.1) having the sequence of SEQ ID NO: 28). As used herein, apolypeptide that is homologous to an E. coli protein has a polypeptidesequence with at least about 90%, at least about 95%, at least about97%, at least about 98%, or at least about 99% sequence homology withthe polypeptide sequence of the E. coli protein.

In certain embodiments, the protein involved in lysine biosynthesis isone or more of aspartate kinase (LysC), dihydrodipicolinate synthase(DapA), diaminopimelate decarboxylase (LysA), a fragment, and/or mutantthereof. In certain embodiments, the protein involved in lysinebiosynthesis is from the genera Escherichia. In certain embodiments, theprotein involved in lysine biosynthesis is from the species E. coli. Forexample, the protein may be E. coli aspartate kinase (LysC or AKIII)protein (SEQ ID NO: 3), which is encoded by the polynucleotide sequenceof the lysC gene. In some embodiments, the protein may be E. colidihydrodipicolinate synthase (DapA or DHDPS) protein (SEQ ID NO: 4),which is encoded by the polynucleotide sequence of the dapA gene. Incertain embodiments, the protein may be E. coli diaminopimelatedecarboxylase (LysA) protein (SEQ ID NO: 5), which is encoded by thepolynucleotide sequence of the lysA gene. In certain embodiments, theprotein involved in lysine biosynthesis is one or more proteins listedin Table 2, fragments thereof and/or mutants thereof.

In certain embodiments, the first expression plasmid vector may be usedfor the production of a lysine derived product as described herein. Incertain embodiments, a lysine derived product may be cadaverine asdescribed herein.

In certain embodiments, the second, third and/or fourth polynucleotidemay be a recombinant or non-naturally occurring polynucleotide. Incertain embodiments, the second polynucleotide may be cDNA. In certainembodiments, the second, third and/or fourth polynucleotide may beobtained by codon optimization for optimal polypeptide expression in aparticular microorganism (e.g., E. coli, H. alvei, or P. aeruginosa).

Another aspect of the invention relates to a transformant comprising oneor more first expression plasmid vectors that are the same or different,and disclosed herein in a host cell.

The first expression plasmid vectors; host cell; backbone plasmid; andfurther additions to the first expression plasmid vector are the same asdescribed supra.

As used herein, a transformant is a host cell that has been altered byintroducing one or more expression plasmid vectors in the host cell,wherein the one or more expression plasmid vectors are the same ordifferent. In certain embodiments, the transformant is obtained byintroducing an expression plasmid vector through transformation into ahost cell displaying competence to the plasmid vector.

In certain embodiments, the transformant may be used for the productionof lysine or a lysine derived product as described herein. In certainembodiments, a lysine derived product may be cadaverine as describedherein.

Another aspect of the invention relates to a transformant comprising oneor more first expression plasmid vectors that are the same or differentand disclosed herein in a host cell, the transformant furthercomprising, consisting, or consisting essentially of:

-   -   one or more second expression plasmid vectors that are the same        or different, and each comprises, consists, or consists        essentially of:        -   one or more fifth polynucleotides that are the same or            different and independently selected from the group            consisting of a first polynucleotide encoding a first            polypeptide comprising, consisting, or consisting            essentially of a tetracycline efflux pump polypeptide, a            fragment thereof or a mutant thereof, a third polynucleotide            encoding a third polypeptide comprising, consisting, or            consisting essentially of a lysine decarboxylase            polypeptide, a fragment thereof or a mutant thereof, and a            fourth polynucleotide encoding a fourth polypeptide            comprising, consisting, or consisting essentially of a            lysine biosynthesis polypeptide, a fragment thereof or a            mutant thereof; and    -   a backbone plasmid capable of autonomous replication in a host        cell,    -   wherein the one or more first and second expression plasmid        vectors are used for production of lysine or a lysine-derived        product.

The first expression plasmid vectors; first polynucleotides, fragmentsand mutants thereof; first polypeptides, fragments and mutants thereof;tetracycline efflux pump polypeptides, fragments and mutants thereof;third polynucleotides, fragments and mutants thereof; thirdpolypeptides, fragments and mutants thereof; lysine decarboxylasepolypeptides, fragments and mutants thereof; fourth polynucleotides,fragments and mutants thereof; fourth polypeptides, fragments andmutants thereof; lysine biosynthesis polypeptides, fragments and mutantsthereof; host cell; backbone plasmid; and further additions to the firstexpression plasmid vector are the same as described supra.

In certain embodiments, the second expression plasmid vector furthercomprises one or more sixth polynucleotides that are the same ordifferent and each encodes a sixth polypeptide comprising, consistingof, or consisting essentially of an antibiotic resistance protein, afragment thereof, or a mutant thereof. In certain embodiments, theantibiotic resistance protein may be a tetracycline resistance proteinor an oxytetracycline-resistance protein. In certain embodiments, theantibiotic resistance protein may be selected from the group thatcomprises, consists of, or consists essentially of OtrA, OtrB, OtrC,Tcr3, a fragment, and/or mutant thereof. In certain embodiments, OtrAmay be from the genus Mycobacterium or Streptomyces. In certainembodiments, OtrB, OtrC, and Tcr3 may be from the genus Streptomyces. Incertain embodiments, the sixth polynucleotide may be selected from thegroup that comprises, consists of, or consists essentially of otrA,otrB, otrC, tcr3, a fragment, or mutant thereof. In certain embodiments,otrA may be from the genus Mycobacterium or Streptomyces. In certainembodiments, otrB, otrC, and tcr3 may be from the genus Streptomyces.

In certain embodiments, the transformant may be used for the productionof lysine or a lysine derived product as described herein. In certainembodiments, a lysine derived product may be cadaverine as describedherein.

Another aspect of the invention relates to a mutant host cellcomprising, consisting of, or consisting essentially of:

one or more first polynucleotides that are the same or different, andintegrated into a chromosome of a host cell, wherein each of the one ormore first polynucleotides encodes one or more first polypeptides thatare the same or different and each comprises, consists of, or consistsessentially of a tetracycline efflux pump polypeptide, a fragmentthereof, or a mutant thereof.

The first polynucleotides, fragments and mutants thereof; firstpolypeptides, fragments and mutants thereof; tetracycline efflux pumppolypeptides, fragments and mutants thereof; and host cell are the sameas described supra.

In certain embodiments, the mutant host cell may be used for theproduction of lysine or a lysine derived product as described herein. Incertain embodiments, a lysine derived product may be cadaverine asdescribed herein.

In certain embodiments, the first polynucleotide may be integrated intothe host cell chromosome according to the PCR-mediated gene replacementmethod (see, e.g. Datsenko, 2000 for an overview of the PCR-mediatedgene replacement method, which is incorporated herein by reference inits entirety). Integrated chromosomes may also be produced by othersuitable methods.

Another aspect of the invention relates to a mutant host cellcomprising, consisting of, or consisting essentially of:

one or more first polynucleotides integrated into a chromosome of a hostcell, wherein the one or more first polynucleotides are the same ordifferent, and each of the one or more first polynucleotides encodes oneor more first polypeptides that are the same or different and eachcomprises, consists of, or consists essentially of a tetracycline effluxpump polypeptide, a fragment thereof, or a mutants thereof;

one or more second polynucleotides integrated into a chromosome of thehost cell, wherein the one or more second polynucleotide are the same ordifferent and independently selected from the group consisting of:

-   -   a third polynucleotide encoding a third polypeptide comprising,        consisting of, or consisting essentially of a lysine        decarboxylase polypeptide, a fragment thereof or a mutant        thereof, and    -   a fourth polynucleotide encoding a fourth polypeptide        comprising, consisting of, or consisting essentially of a lysine        biosynthesis polypeptide, a fragment thereof or a mutant        thereof.

The first polynucleotides; first polypeptides; tetracycline efflux pumppolypeptides, fragments and mutants thereof; second polynucleotides;third polynucleotides; third polypeptides; lysine decarboxylasepolypeptides, fragments and mutants thereof; fourth polynucleotides;fourth polypeptides; lysine biosynthesis polypeptides, fragments andmutants thereof; and host cell are the same as described supra.

In certain embodiments, when there are a plurality of the firstpolypeptides, each first polypeptide may be the same or different andmay be expressed individually or as a fusion protein.

In certain embodiments, when there are a plurality of the secondpolynucleotides, each third polypeptide may be the same or different,and each fourth polypeptide may be the same or different; the third andfourth polypeptides may be expressed individually or as a fusionprotein.

In certain embodiments, the first and second polynucleotides may beintegrated into the host cell chromosome according to the PCR-mediatedgene replacement method (see, e.g. Datsenko, 2000 for an overview of thePCR-mediated gene replacement method, which is incorporated herein byreference in its entirety). Integrated chromosomes may also be producedby other suitable methods.

In certain embodiments, the mutant host cell may be used for theproduction of lysine or a lysine derived product as described herein. Incertain embodiments, a lysine derived product may be cadaverine asdescribed herein.

Another aspect of the invention relates to a method for producing lysinecomprising:

obtaining a transformant and/or mutant host cell as disclosed herein;

culturing the transformant and/or mutant host cell under conditionseffective for the expression of the lysine; and

harvesting the lysine.

In certain embodiments, the transformant and/or mutant host cell may beany of those as described herein. For example, the transformant used toproduce lysine may be obtained by transforming one or more expressionplasmid vectors that are the same or different, and disclosed hereininto a host cell.

The transformant and/or mutant host cell may be cultured using a mediumcontaining carbon sources and non-carbon nutrient sources. Examples ofcarbon sources include, without limitation, sugar (e.g. carbohydratessuch as glucose and fructose), oil and/or fat, fatty acid, and/orderivatives thereof. The oil and fat may contain saturated and/orunsaturated fatty acids having 10 or more carbon atoms, e.g. coconutoil, palm oil, palm kernel oil, and the like. The fatty acid may be asaturated and/or unsaturated fatty acid, e.g. hexanoic acid, octanoicacid, decanoic acid, lauric acid, oleic acid, palmitic acid, linoleicacid, linolenic acid, myristic acid, and the like. Examples ofderivatives of a fatty acid include, without limitation, esters andsalts thereof. Examples of non-carbon sources include, withoutlimitation, nitrogen sources, inorganic salts, and other organicnutrient sources.

For example, a medium may contain a carbon source assimilable by thetransformant and/or mutant host cell, optionally with one or more othersources independently selected from the group consisting of a nitrogensource, an inorganic salt and another organic nutrient source. Incertain embodiments, the weight percentage of the nitrogen source isabout 0.01 to about 0.1% of the medium. Examples of the nitrogen sourcemay comprise ammonia, ammonium salts (e.g. ammonium chloride, ammoniumsulfate and ammonium phosphate), peptone, meat extract, yeast extract,and the like. Examples of the inorganic salts include, withoutlimitation, potassium dihydrogen phosphate, dipotassium hydrogenphosphate, magnesium phosphate, magnesium sulfate, sodium chloride, andthe like. Examples of the other organic nutrient source include, withoutlimitation, amino acids (e.g. glycine, alanine, serine, threonine andproline), vitamins (e.g. vitamin B1, vitamin B12 and vitamin C), and thelike.

The culture may be carried out at any temperature as long as the cellscan grow, and preferably at about 20° C. to about 40° C., or about 35°C. The culture period may be about 1, about 2, about 3, about 4, about5, about 6, about 7, about 8, about 9, or about 10 days.

In one embodiment, the transformant and/or mutant host cell is culturedin a medium containing peptides, peptones, vitamins (e.g. B vitamins),trace elements (e.g. nitrogen, sulfur, magnesium), and minerals.Examples of such medium include, without limitation, commonly knownLysogeny broth (LB) mediums comprising tryptone, yeast extract and NaClsuspended in water (e.g. distilled or deionized).

Another aspect of the invention relates to a method for producingcadaverine (1,5-pentanediamine) comprising, consisting of, or consistingessentially of:

1a) cultivating the transformant and/or mutant host cell as disclosedherein,

1b) producing cadaverine using the culture obtained from step 1a todecarboxylate lysine, and

1c) extracting and purifying cadaverine using the culture obtained fromstep 1 b.

In certain embodiments, the transformant and/or mutant host cell may beany of those as described herein.

Cultivating the transformant may comprise the steps of culturing thetransformant as described supra.

As used herein, “using the culture obtained from step 1a” may comprisefurther processes of the culture obtained from step 1a. For example,using a buffer solution to dilute the culture; centrifuging the cultureto collect the cells; resuspending the cells in a buffer solution; orlysing the cells into cell lysate; or/and purifying lysine decarboxylasefrom the cell lysate.

In another embodiment, step 1c of the method further comprises thefollowing steps:

1d) separating the solid and liquid components of the reaction obtainedfrom step 1b;

1e) adjusting the pH of the liquid component obtained from step 1d toabout 14 or higher;

1f) removing water from the liquid component obtained from step 1e; and

1g) recovering cadaverine.

In step 1d, the separation of the solid and liquid components of thereaction of step 1b may be accomplished by conventional centrifugationand/or filtration.

In step 1e, the pH of the liquid component of step 1d may be adjusted byadding a base, e.g. NaOH. NaOH may be added as a solid and/or a solution(e.g. an aqueous solution).

In step 1f, the water may be removed by distillation at ambient pressureor under vacuum.

In step 1g, cadaverine may be recovered by distillation at ambientpressure or under vacuum.

Another aspect of the invention relates to biobased cadaverine preparedaccording to the method disclosed herein.

As used herein, a “biobased” compound means the compound is consideredbiobased under Standard ASTM D6866.

Another aspect of the invention relates to a polyamide having astructure of Structure 1:

-   -   including stereoisomers thereof, wherein:    -   m=4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, or 22;    -   n=4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, or 22;    -   j=about 100˜about 1,000,000; and    -   the polyamide is prepared from one or more diamines having        carbon numbers of m and one or more dicarboxylic acids having        carbon numbers of n, at least one of the diamines and        dicarboxylic acids comprises biobased carbon under Standard ASTM        D6866, and the m or n of each diamine or dicarboxylic acid can        be the same or different.

In one embodiment, the diamine is biobased cadaverine, more preferablybiobased cadaverine prepared according to the method disclosed herein.Examples of the dicarboxylic acids include, without limitation,C₁₀dicarboxylic acid, C₁₁dicarboxylic acid, C₁₂dicarboxylic acid,C₁₃dicarboxylic acid, C₁₄dicarboxylic acid, C₁₆dicarboxylic acid,C₁₈dicarboxylic acid, and any combinations thereof. In certainembodiments, all or part of the C_(n)dicarboxylic acids are biobased.

In another embodiments, the polyamide has a structure described above,wherein:

-   -   the polyamide is formed by reacting biobased cadaverine with one        or more dicarboxylic acids, more preferably the biobased        cadaverine is prepared according to the method disclosed herein;    -   n=4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, or 22;    -   j=about 100˜about 1,000,000, about 1000˜about 100,000, or about        1000˜about 10,000; and    -   the dicarboxylic acids comprise biobased carbon under Standard        ASTM D6866.

Another aspect of the invention relates to a method of making thepolyamides disclosed herein comprising preparing biobased cadaverine asthe Cmdiamine according to the method disclosed herein.

In one embodiment, the method further comprises preparing one or morebiobased Cndicarboxylic acids.

In another embodiment, the method further comprises preparing thepolyamide by reacting biobased cadaverine with one or more biobasedCndicarboxylic acids.

Another aspect of the invention relates to a composition comprising oneor more polyamides disclosed herein.

In one embodiment, the diamine is biobased cadaverine, more preferablybiobased cadaverine prepared according to the method disclosed herein.Examples of the dicarboxylic acids include, without limitation,C10dicarboxylic acid, C11dicarboxylic acid, C12dicarboxylic acid,C13dicarboxylic acid, C14dicarboxylic acid, C16dicarboxylic acid,C18dicarboxylic acid, and any combinations thereof. In certainembodiments, all or part of the Cndicarboxylic acids are biobased.

In another embodiment, the polyamide has a structure described above,wherein:

-   -   the polyamide is formed by reacting biobased cadaverine with one        or more dicarboxylic acids, more preferably the biobased        cadaverine is prepared according to the method disclosed herein;    -   n=4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, or 22;    -   j=about 100˜about 1,000,000, about 1000˜about 100,000, or about        1000˜about 10,000; and    -   the dicarboxylic acids comprise biobased carbon under Standard        ASTM D6866.

Another aspect of the invention relates to a method of preparing1,5-diisocyanatopentane comprising:

-   -   2a) preparing biobased cadaverine as disclosed herein; and    -   2b) converting biobased cadaverine obtained from step 2a to        1,5-diisocyanatopentane.

Step 2b may comprise using any known method to convert diamine intoisocyanate. An example of said method is the traditional phosgenemethod, which includes one-step high temperature phosgene method (i.e.mixing phosgene with diamine at high temperature to obtain isocyanate),the improved two-step phosgene method, and the triphosgene method inwhich triphosgene is used instead of phosgene. There are also othermethods that do not use phosgene as a raw material. An example of saidmethod is hexanediamine carbonylation which uses CO2 instead ofphosgene: CO2 is added into a solution of a primary amine and an organicbase, then a proper amount of phosphorus electrophilic reagents is addedinto the reaction solution to start an exothermic dehydration reactionto obtain isocyanate. Another example is carbamate thermal decompositionmethod wherein a primary amine is converted to a carbamate, and then thecarbamate is heated to decompose and generate isocyanate.

The abbreviations used for the amino acids, polypeptides, basesequences, and nucleic acids are based on the abbreviations specified inthe IUPAC-IUB Communication on Biochemical Nomenclature, Eur. J.Biochem., 138:9 (1984), “Guideline for Preparing SpecificationsIncluding Base Sequences and Amino Acid Sequences” (United States Patentand Trademark Office), and those commonly used in this technical field.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words “comprise,” “comprising,” and thelike are to be construed in an inclusive sense (i.e., to say, in thesense of “including, but not limited to”), as opposed to an exclusive orexhaustive sense. The words “herein,” “above,” “below,” “supra,” andwords of similar import; when used in this application, refer to thisapplication as a whole and not to any particular portions of thisapplication. Where the context permits, words in the above DetailedDescription using the singular or plural number may also include theplural or singular number respectively. The words “or,” and “and/or” inreference to a list of two or more items, covers all of the followinginterpretations of the word: any of the items in the list, all of theitems in the list, and any combination of the items in the list.

In E. coli, lysine is known to be transported across the membrane intothe cell through three pathways: one mediated by the lysine-specificpermease LysP, another mediated by the ArgT ABC transporter that alsorecognizes arginine and ornithine, and the third is mediated by thelysine/cadaverine antiporter, CadB. Bacteria use pumps to transportantibiotics out of the cell and nutrients into a cell. Tetracyclines areantibiotics that stop protein synthesis and inhibit cell growth. ManyGram-positive and Gram-negative bacteria have evolved to expressproteins that pump tetracycline out of the cell. To date, sixty-onetetracycline resistance genes have been sequenced in bacteria thatproduce or do not produce tetracyclines. The most commonly usedtetracycline efflux pump in the laboratory is TetA, a protein thatlocalizes to the inner membrane and catalyzes the exchange ofmonocationic magnesium-tetracycline chelate complex from inside the cellwith a proton from outside the cell.

A mechanism that enables E. coli transport of lysine and lysine-derivedproducts across the membrane is disclosed herein. As shown in theExamples below, the increased expression of tetracycline efflux pumpprotein, TetA, resulted in an increased production of lysine in E. coli.Additionally, the increased expression of TetA in both E. coli and H.alvei resulted in a higher yield of cadaverine, a metabolite derivedfrom lysine. Therefore, the data provided herein indicate thattetracycline efflux pumps can be used to increase the yield of lysineand/or lysine derived products.

The following examples are intended to illustrate various embodiments ofthe invention. As such, the specific embodiments discussed are not to beconstrued as limitations on the scope of the invention. It will beapparent to one skilled in the art that various equivalents, changes,and modifications may be made without departing from the scope ofinvention, and it is understood that such equivalent embodiments are tobe included herein. Further, all references cited in the disclosure arehereby incorporated by reference in their entirety, as if fully setforth herein.

EXAMPLES Example 1. Construction of Strains Containing Plasmid Vectorsthat Encode a Tetracycline Efflux Pump

The E. coli gene, tetA (SEQ ID NO: 1), that encodes a tetracyclineefflux pump, TetA (SEQ ID NO: 2), was amplified from the E. coli cloningvector pBR322 using the PCR primers tetA-F and tetA-R (FIG. 1). Theamplified DNA was digested with the restriction enzymes SacI and XbaI,and ligated into a pUC18 plasmid vector and a pCIB10 plasmid vector tocreate pCIB17 and pCIB20, respectively (FIG. 2). pCIB17 was transformedinto E. coli and H. alvei to create strains CIB17-EC and CIB17-HA,respectively (FIG. 2).

Example 2. Construction of Strains Containing Plasmid Vectors thatEncode Either Wild-Type CadA or an Inactive CadA Mutant

A plasmid vector containing wild-type E. coli cadA, which encodes thelysine decarboxylase CadA (SEQ ID NO: 6), was constructed by cloningwild-type cadA into pUC18 to generate the positive control, pCIB60 (FIG.2). A plasmid vector containing an inactive mutant cadA was constructedby truncating the C-terminus of cadA by introducing a premature stopcodon which resulted in a mutation at amino acid 566 of CadA and atruncated CadA protein (CadA (aa1-565) (where “aa” is amino acids) (SEQID NO: 33). The primers cadAm1-F and cadAm1-R (FIG. 1) were used toamplify a fragment of cadA. The amplified mutant cadA and the pCIB60plasmid were digested using the restriction enzymes XhoI and SphI. Thedigested mutant cadA was then ligated into the digested pCIB60 plasmidvector to replace the wild type cadA with the mutant cadA. The plasmidvector expressing the truncated cadA gene was designated as pCIB63 (FIG.2). pCIB60 was transformed into E. coli and H. alvei to create thestrains CIB60-EC and CIB60-HA, respectively. pCIB63 was transformed intoE. coli and H. alvei to create the strains CIB63-EC and CIB63-HA,respectively (FIG. 2).

Example 3. Production of Cadaverine from E. coli and H. Alvei StrainsExpressing a Recombinant Tetracycline Efflux Pump

A single colony of each strain was grown overnight in LB medium withampicillin (100 μg/mL) in a 2.5 mL culture at 29° C. E. coli and H.alvei transformed with the empty vector, pUC18, were used as negativecontrols. The following day, 2.5 mL of minimal media was supplementedwith ampicillin (100 μg/mL), and lysine-HCl and PLP to a finalconcentration of 20 g/L and 0.1 mM, respectively. Each culture wasincubated at 37° C. for 5 hours and 21 hours. 1 mL of sample was takenfrom each culture at the 5- and 21-hour time points to quantifycadaverine production using nuclear magnetic resonance (NMR). Cadaverineproduction at both time points from strains expressing the mutant, CadA(aa1-565), wild-type CadA or TetA is presented in Table 3.

TABLE 3 Production of cadaverine in E. coli and H. alvei by expressionof CadA or TetA. 5 hrs 21 hrs Host Strain Enzyme(s) Gene(s) (g/kg)(g/kg) E. coli none none 0.20 N.A. CIB63-EC CadA (aa1-565) mutant cadA 2.99 ± 0.005 3.78 ± 0.04 CIB60-EC CadA cadA 8.93 ± 0.9 11.7 ± 0.02CIB17-EC TetA tetA 5.14 ± 0.1 11.1 ± 0.5  H. alvei none none 1.27 N.A.CIB63-HA CadA (aa1-565) mutant cadA 0.24 N.A. CIB60-HA CadA cadA 9.64 ±0.3 N.A. CIB17-HA TetA tetA 9.28 ± 1.8 N.A. N.A.: data not available

As indicated in Table 3, expression of TetA in E. coli resulted in ahigher yield of cadaverine production as compared to the negativecontrol (no enzyme) and the mutant CadA (aa1-565) (5 hours: 5.14 g/kgcompared to 0.20 g/kg and 2.99 g/kg, respectively; 21 hours: 11.1 g/kgcompared to N.A. and 3.78 g/kg). Also, expression of TetA in H. alveiresulted in a higher yield of cadaverine production as compared to thenegative control (none) and the mutant CadA (aa1-565) (5 hours: 9.28g/kg compared to 1.27 g/kg and 0.24 g/kg, respectively).

Example 4. Production of Cadaverine from E. coli Strains Co-Expressing aRecombinant Lysine Decarboxylase and a Recombinant Tetracycline EffluxPump

The tetA gene was cloned into the pCIB60 plasmid vector behind the cadAgene. First, tetA was amplified from pBR322 using primers tetA-F2 andtetA-R2 (FIG. 1). Next, the amplified PCR product and pCIB60 plasmidvector were digested using the restriction enzymes XbaI and HindIII, andthe products were ligated together to create pCIB77. pCIB77 wastransformed into E. coli MG1655 K12 to create the strain CIB77 (FIG. 2).

A single colony of each strain was grown overnight in LB medium withampicillin (100 μg/mL) in a 2.5 mL culture at 29° C. The following day,minimal media was supplemented with ampicillin (100 μg/mL), andlysine-HCl and PLP to a final concentration of 20 g/L and 0.1 mM,respectively. Each culture was incubated at 37° C. for 3 hours and 6hours. 1 mL of sample was taken from each culture at the 3- and 6-hourtime points to quantify cadaverine production using NMR. Cadaverineproduction at both time points from strains expressing CadA or TetA andCadA is presented in Table 4.

TABLE 4 Production of cadaverine in E. coli by expression of CadA orCadA and TetA. Host Strain Enzyme(s) Gene(s) 3 hrs (g/kg) 6 hrs (g/kg)None None 0.14 0.31 E. coli CIB60 CadA cadA 4.38 ± 0.65 6.20 ± 0.61CIB77 TetA, CadA tetA, cadA 5.54 ± 1.30 8.06 ± 0.65

Table 4 indicates that expression of TetA and CadA together resulted ina higher yield of cadaverine production in E. coli when compared toexpression of only CadA (3 hours: 5.54 g/kg compared to 4.38 g/kg,respectively; 6 hours: 8.06 g/kg compared to 6.20 g/kg, respectively).

Example 5: Production of Lysine from Strains Co-Expressing a RecombinantTetracycline Efflux Pump and Recombinant Proteins (LysC, DapA, LysA)from the Lysine Biosynthetic Pathway

Three genes from E. coli, lysC, dapA, and lysA, encode proteins involvedin the E. coli lysine biosynthetic pathway: aspartate kinase (LysC orAKIII, encoded by lysC), dihydrodipicolinate synthase (DapA or DHDPS,encoded by dapA), and diaminopimelate decarboxylase (LysA, encoded bylysA). The three genes were cloned into a plasmid vector and the threeproteins, LysC (SEQ ID NO: 3), DapA (SEQ ID NO: 4), and LysA (SEQ ID NO:5) were overexpressed in E. coli. The gene lysC was amplified from theE. coli MG1655 K12 genomic DNA using the primers lysC-F and lysC-R (FIG.1), and the amplified fragment was digested using SacI and BamHI, andligated into pUC18 to create pCIB7 (FIG. 2). The gene dapA was amplifiedfrom the E. coli MG1655 K12 genomic DNA using the primers dapA-F anddapA-R (FIG. 1), and the amplified fragment was digested using BamHI andXbaI, and ligated into pCIB7 to create pCIB8 (FIG. 2). The gene lysA wasamplified from the E. coli MG1655 K12 genomic DNA using the primerslysA-F and lysA-R (FIG. 1), and the amplified fragment was digestedusing XbaI and Sail, and ligated into pCIB8 to create pCIB9. Thethree-gene operon was amplified from pCIB9 using the primers lysC-F andlysA-R (FIG. 1). The amplified product was digested using SacI and SalI,and the digested fragment was ligated into pCIB10 to create pCIB32 (FIG.2). pCIB32 was transformed into E. coli to create the strain CIB32.CIB32 was further transformed with pCIB20, the plasmid containing thetetA gene.

A single colony was grown overnight at 37° C. in 3 mL of mediumcontaining 4% glucose, 0.1% KH₂PO₄, 0.1% MgSO₄, 1.6% (NH₄)₂SO₄, 0.001%FeSO₄, 0.001% MnSO₄, 0.2% yeast extract, 0.05% L-methionine, 0.01%L-threonine, 0.005% L-isoleucine, ampicillin (100 μg/mL), andtetracycline (10 μg/mL). The following day, each culture was inoculatedinto 100 mL of fresh medium with ampicillin (100 μg/mL) and tetracycline(10 μg/mL), and grown for 21 hours at 37° C., at which point theconcentration of lysine in each culture was determined (Table 5).

TABLE 5 Production of lysine in E. coli by expression of TetA andproteins from the lysine biosynthetic pathway. Lysine Host Strain andPlasmid Enzyme(s) Gene(s) (mg/g) E. coli MG1655 K12 none none n.d. CIB32LysC, DapA, lysC, dapA, 1.47 ± 0.05 LysA lysA CIB32 + pCIB20 LysC, DapA,lysC, dapA, 2.46 ± 0.03 LysA, TetA lysA, tetA n.d.: none detected

As indicated in Table 5, the expression of TetA along with proteinsinvolved in the lysine biosynthetic pathway (i.e., CIB32+pCIB20)resulted in a higher yield of lysine as compared to expression ofproteins involved in the lysine biosynthetic pathway without TetA (i.e.,CIB32) (compare 2.46 mg/g to 1.47 mg/g).

Example 6: Production of Lysine from Strains Co-Expressing a RecombinantTetracycline Efflux Pump and Recombinant Proteins (LysC, DapA, LysA,DapB, DapD, AspC, Asd) from the Lysine Biosynthetic Pathway

Next, the expression of four additional genes, asd, dapB, dapD, andaspC, which are involved in the lysine biosynthetic pathway of E. coli,was enhanced. These genes encode the following enzymes: aspartatesemialdehyde dehydrogenase (Asd (SEQ ID NO: 22), encoded by asd),dihydrodipicolinate reductase (DapB or DHDPR (SEQ ID NO: 23), encoded bydapB), tetrahydrodipicolinate succinylase (DapD (SEQ ID NO: 24), encodedby dapD), and aspartate transaminase (AspC (SEQ ID NO: 25), encoded byaspC). The gene asd was amplified from the E. coli MG1655 K12 genomicDNA using the primers asd-F and asd-R (FIG. 1), and the amplifiedfragment was digested using SacI and BamHI, and ligated into pUC18 tocreate pCIB12 (FIG. 2). The gene dapB was amplified from the E. coliMG1655 K12 genomic DNA using the primers dapB-F and dapB-R (FIG. 1), andthe amplified fragment was digested using BamHI and XbaI, and ligatedinto pCIB12 to create pCIB13 (FIG. 2). The gene dapD was amplified fromthe E. coli MG1655 K12 genomic DNA using the primers dapD-F and dapD-R(FIG. 1), and the amplified fragment was digested using XbaI and Sail,and ligated into pCIB13 to create pCIB14 (FIG. 2). Similarly, the geneaspC was amplified from the E. coli MG1655 K12 genomic DNA using theprimers aspC-F and aspC-R (FIG. 1), and the amplified fragment wasdigested using XbaI and Sail, and ligated into pCIB13 to create pCIB31(FIG. 2). The gene tetA was amplified from pCIB20 using the primerstetA-F3 and tetA-R3 (FIG. 1), and the amplified fragment was digestedusing XhoI and SphI and ligated into pCIB14 and pCIB31 to generateplasmids pCIB15 and pCIB59, respectively (FIG. 2). pCIB59 and pCIB15were each transformed into strain CIB32.

A single colony was grown overnight at 37° C. in 3 mL of mediumcontaining 4% glucose, 0.1% KH₂PO₄, 0.1% MgSO₄, 1.6% (NH₄)₂SO₄, 0.001%FeSO₄, 0.001% MnSO₄, 0.2% yeast extract, 0.05% L-methionine, 0.01%L-threonine, 0.005% L-isoleucine, ampicillin (100 μg/mL), andtetracycline (10 μg/mL). The following day, each culture was inoculatedinto 100 mL of fresh medium with ampicillin (100 μg/mL) and tetracycline(10 μg/mL), and grown for 21 hours at 37° C., at which point theconcentration of lysine in each culture was determined (Table 6).

TABLE 6 Production of lysine in E. coli by expression of TetA andproteins from the lysine biosynthetic pathway Strain and Lysine HostPlasmid Enzyme(s) Gene(s) (mg/g) E. MG1655 none none n.d. coli K12 CIB32LysC, DapA, LysA lysC, dapA, lysA 0.61 ± 0.02 CIB32 + LysC, DapA, LysA,lysC, dapA, lysA, 1.03 ± 0.01 pCIB20 TetA tetA CIB32 + LysC, DapA, LysA,lysC, dapA, lysA, 1.16 ± 0.03 pCIB15 Asd, DapB, DapD, asd, dapB, dapD,TetA tetA CIB32 + LysC, DapA, LysA, lysC, dapA, lysA, 1.14 ± 0.02 pCIB59Asd, DapB, AspC, asd, dapB, aspC, TetA tetA n.d.: none detected

As indicated in Table 6, the expression of TetA along with proteinsinvolved in the lysine biosynthetic pathway (i.e., CIB32+pCIB20,CIB32+pCIB15, and CIB32+pCIB59) resulted in a higher yield of lysine ascompared to expression of proteins involved in the lysine biosyntheticpathway without TetA (i.e., CIB32) (compare 1.03 mg/g, 1.16 mg/g, and1.14 mg/g to 0.61 mg/g).

Example 7: Production of Lysine from Strains Co-Expressing a RecombinantTetracycline Efflux Pump and Various Aspartokinases

Various aspartokinases were expressed in order to increase lysineproduction. Two pairs of mutations were chosen that enabled the E. coliaspartokinase III (LysC or AKIII, encoded by lysC, SEQ ID NO: 3) to havean increased feedback resistance to lysine. The gene encoding the firstmutant, LysC-1 (M318I, G323D) (SEQ. ID NO: 26) was constructed using theprimers 318-F, 318-R, 323-F, 323-R (FIG. 1). The gene encoding thesecond mutant, LysC-2 (T344M, T352I) (SEQ. ID NO: 27), was constructedusing the primers 344-F, 344-R, 352-F, 352-R (FIG. 1). The genesencoding LysC-1 (M318I, G323D) and LysC-2 (T344M, T352I) were clonedinto pCIB32 and replaced the wild-type E. coli aspartokinase, LysC, tocreate the plasmids pCIB43 and pCIB44, respectively (FIG. 2). Theaspartokinase from Streptomyces strains that is capable of producingpolylysine was previously suggested, but not proven, to be more feedbackresistant to lysine compared to E. coli aspartokinase. As such, theaspartokinase gene from Streptomyces lividans was codon optimized,synthesized, and cloned in place of wild-type lysC in pCIB32 in order tocreate the plasmid pCIB55 using the primers SlysC-F and SlysC-R (FIG.1). The resulting aspartokinase protein that was expressed was namedS-LysC (SEQ ID NO: 28). pCIB43, pCIB44, and pCIB55 were transformed intoE. coli MG1655 to create the strains CIB43, CIB44, and CIB55,respectively (FIG. 2). pCIB59 was also transformed into CIB55.

A single colony was grown overnight at 37° C. in 3 mL of mediumcontaining 4% glucose, 0.1% KH₂PO₄, 0.1% MgSO₄, 1.6% (NH₄)₂SO₄, 0.001%FeSO₄, 0.001% MnSO₄, 0.2% yeast extract, 0.05% L-methionine, 0.01%L-threonine, 0.005% L-isoleucine, ampicillin (100 μg/mL), andtetracycline (10 μg/mL). The following day, each culture was inoculatedinto 100 mL of fresh medium with ampicillin (and tetracycline in thecase of CIB55+pCIB59), and grown for 63 hours, at which point theconcentration of lysine in each culture was determined.

TABLE 7 Production of lysine in E. coli by expression of TetA andproteins from the lysine biosynthetic pathway. Strain and Lysine HostPlasmid Enzyme(s) Gene(s) (mg/g) E. coli MG1655 K12 none none n.d. CIB32LysC, DapA, LysA lysC, dapA, 0.48 ± 0.01 lysA CIB43 LysC-1 (M318I,G323D), lysC-1, dapA, 1.57 ± 0.02 DapA, LysA lysA CIB44 LysC-2 (T344M,T352I), lysC-2, dapA, 1.50 ± 0.02 DapA, LysA lysA CIB55 S-LysC, DapA,LysA S-lysC, dapA, 1.55 ± 0.02 lysA CIB55 + S-LysC, DapA, LysA, Asd,S-lysC, dapA, 1.66 ± 0.01 pCIB59 DapB, AspC, TetA lysA, asd, dapB, aspC,tetA n.d. = not detected

Example 8: Production of Lysine in E. coli

The two lysine operons consisting of the genes S-lysC, dapA, lysA, asd,dapB, and aspC were combined into a single vector. The operon frompCIB55 consisting of the genes S-lysC, dapA, and lysA was amplifiedusing the primers SAL-F and SAL-R (FIG. 1). The operon from pCIB31consisting of the genes asd, dapB, and aspC was amplified using theprimers ABC-F and ABC-R (FIG. 1). The operon from pCIB59 consisting ofthe genes asd, dapB, and aspC and the tetA gene was amplified using theprimers ABC-F and ABCT-R (FIG. 1). The products were digested using therestriction enzymes ApaI and KpnI. The digested products of pCIB55 andpCIB31 were ligated to form pCIB92 and the digested products of pCIB55and pCIB59 were ligated to form pCIB103 (FIG. 2).

E. coli was used as a host to produce lysine. The plasmids, pUC18,pCIB92, and pCIB103 were independently co-transformed with pCIB110 intoE. coli. Two colonies from each transformation were grown overnight at37° C. in 3 mL of LB medium supplemented with ampicillin (100 mg/L) for18 hours. The following day, each culture was assayed for cadaverineusing NMR. The lysine production is shown in Table 8.

TABLE 8 Production of cadaverine in E. coli by expression of TetA andproteins from the lysine biosynthetic pathway Lysine Host PlasmidsEnzyme(s) Gene(s) (mg/g) E. coli pUC18 none none n.d. pCIB92 S-LysC,DapA, S-lysC, dapA, 1.62 ± 0.02 LysA, Asd, DapB, lysA, asd, AspC dapB,aspC pCIB103 S-LysC, DapA, S-lysC, dapA, 1.70 ± 0.01 LysA, Asd, DapB,lysA, asd, AspC, TetA dapB, aspC, tetA n.d.: none detected

As indicated in Table 8, the expression of TetA along with proteinsinvolved in the lysine biosynthetic pathway (i.e., pCIB103) resulted ina higher yield of cadaverine as compared to expression of proteinsinvolved in the lysine biosynthetic pathway without TetA (i.e., pCIB92)(compare 1.70±0.01 g/kg to 1.62±0.02 g/kg).

Example 9: Production of Cadaverine in H. Alvei

H. alvei was used as a host to produce cadaverine without adding anyadditional lysine to the cell. The plasmids, pUC18, pCIB92, and p103were independently transformed into H. alvei. Two colonies from eachtransformation were grown overnight at 37° C. in 3 mL of LB mediumsupplemented with ampicillin (100 mg/L) for 18 hours. The following day,each culture was assayed for cadaverine using NMR. The cadaverineproduction is shown in Table 8.

TABLE 9 Production of cadaverine in H. alvei by expression of TetA andproteins from the lysine biosynthetic pathway Cadaverine Host PlasmidsEnzyme(s) Gene(s) (g/kg) H. alvei pUC18 none none 0.07 ± 0.04 pCIB92S-LysC, DapA, S-lysC, dapA, 0.11 ± 0.005 LysA, Asd, lysA, asd, DapB,AspC dapB, aspC pCIB103 S-LysC, DapA, S-lysC, dapA, 0.36 ± 0.01 LysA,Asd, TetA lysA, asd, DapB, AspC, dapB, aspC, tetA n.d.: none detected

As indicated in Table 8, the expression of TetA along with proteinsinvolved in the lysine biosynthetic pathway (i.e., pCIB103) resulted ina higher yield of cadaverine as compared to expression of proteinsinvolved in the lysine biosynthetic pathway without TetA (i.e., pCIB92)(compare 0.36 g/kg to 0.11 g/kg).

Example 10: Increased Cadaverine Production from Mutant TetA

The full-length TetA protein is 396 amino acids long. Amber mutations(stop codons) were introduced into the tetA polynucleotide sequence inorder to truncate TetA to determine whether the entire TetA protein isnecessary for the increased production of cadaverine. Two truncations ofthe TetA protein were generated. The first truncation, TetA (aa1-185),was generated by using Splicing by Overlap Extension PCR (SOEing PCR)with the primers tetAm1-F, tetAm1-R, tetA-F2, and tetA-R2 (FIG. 1). Thisinserted a T after nucleotide 555 in order to create a premature stopcodon in the tetA polynucleotide sequence (tetA (nt 1-558), where “nt”is nucleotides, SEQ ID NO: 29), which resulted in a mutation at aminoacid 186 and a truncated form of TetA having amino acids 1-185 (SEQ IDNO: 30). The amplified product was cloned into pCIB60 (containing CadA)using the restriction enzymes XbaI and HindIII, and the products wereligated together to create pCIB97 (FIG. 2). The second truncation, TetA(aa1-96), was created using SOEing PCR with the primers tetAm2-F,tetAm2-R, tetA-F2, and tetA-R2 (FIG. 1). This inserted two nucleotides(AA) after nucleotide 289 in order to introduce a premature stop codonin the tetA polynucleotide sequence (tetA (nt 1-291), SEQ ID NO: 31),which resulted in a mutation at amino acid 97 and a truncated form ofTetA having amino acids 1-96 (SEQ ID NO: 32). The amplified product wascloned into pCIB60 to create pCIB98. The plasmids pCIB97 and pCIB98 weretransformed into E. coli MG1655 to create CIB97 and CIB98, respectively(FIG. 2).

A single colony of each strain was grown overnight in LB medium withampicillin (100 μg/mL) in a 2 mL culture at 29° C. The following day,0.9 mL of culture was added to 100 μL of lysine-HCL and 5 μL of PLP to afinal concentration of 40 g/L and 0.1 mM, respectively. Each culture wasincubated at 37° C. for 2 hours. Each sample was quantified forcadaverine production using NMR (Table 9).

TABLE 9 Production of cadaverine in E. coli by expression of TetA andproteins from the lysine biosynthetic pathway Cadaverine Host StrainEnzyme(s) Gene(s) (g/kg) E. coli MG1655 K12 none none n.d. CIB60 CadAcadA 1.1 ± 0.1 CIB77 TetA, CadA tetA, cadA 2.0 ± 1.3 CIB97 TetA (1-185),tetA (nt 1-558), 1.8 ± 0.1 CadA cadA CIB98 TetA (1-96), tetA (nt 1-291),2.6 ± 0.9 CadA cadA n.d.: none detected

As indicated in Table 9, the expression of TetA with CadA and thetruncated forms of TetA with CadA resulted in an increased production ofcadaverine compared with expression of CadA alone (compare 2.0 g/kg(CIB77), 1.8 g/kg (CIB97) and 2.6 g/kg (CIB98) compared to 1.1 g/kg(CIB60)). The amount of cadaverine produced with the expression of bothTetA mutants (TetA (aa1-185) and TetA (aa1-96)) was comparable to theamount of cadaverine produced with the expression of wild type TetA (2.0g/kg), with cells expressing TetA (aa1-96) producing the most cadaverine(2.6 g/kg).

Sequence Listings

SEQ ID NO: 1 (Escherichia coli tetA polynucleotide sequence) ATGAAATCTAACAATGCGCTCATCGTCATCCTCGGCACCGTCACCCTGGATGCTGTAGGCATAGGCTTGGTTATGCCGGTACTGCCGGGCCTCTTGCGGGATATCGTCCATTCCGACAGCATCGCCAGTCACTATGGCGTGCTGCTAGCGCTATATGCGTTGATGCAATTTCTATGCGCACCCGTTCTCGGAGCACTGTCCGACCGCTTTGGCCGCCGCCCAGTCCTGCTCGCTTCGCTACTTGGAGCCACTATCGACTACGCGATCATGGCGACCACACCCGTCCTGTGGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGCATGGCGGCCGACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCGGCATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCATTGGACCGCTGATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGASEQ ID NO: 2 (Escherichia coli TetA polypeptide sequence, encodedby tetA gene)MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHYGVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRIVAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPFLAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIMQLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAIIAGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSLAALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATSTSEQ ID NO: 3 (Escherichia coli aspartokinase III polypeptidesequence, LysC, encoded by lysC gene)MSEIVVSKFGGTSVADFDAMNRSADIVLSDANVRLVVLSASAGITNLLVALAEGLEPGERFEKLDAIRNIQFAILERLRYPNVIREEIERLLENITVLAEAAALATSPALTDELVSHGELMSTLLFVEILRERDVQAQWFDVRKVMRTNDRFGRAEPDIAALAELAALQLLPRLNEGLVITQGFIGSENKGRTTTLGRGGSDYTAALLAEALHASRVDIWTDVPGIYTTDPRVVSAAKRIDEIAFAEAAEMATFGAKVLHPATLLPAVRSDIPVFVGSSKDPRAGGTLVCNKTENPPLFRALALRRNQTLLTLHSLNMLHSRGFLAEVFGILARHNISVDLITTSEVSVALTLDTTGSTSTGDTLLTQSLLMELSALCRVEVEEGLALVALIGNDLSKACGVGKEVFGVLEPFNIRMICYGASSHNLCFLVPGEDAEQVVQKLHSNLFESEQ ID NO: 4 (Escherichia coli dihydrodipicolinate synthasepolypeptide sequence, DapA, encoded by dapA gene)MFTGSIVAIVTPMDEKGNVCRASLKKLIDYHVASGTSAIVSVGTTGESATLNHDEHADVVMMTLDLADGRIPVIAGTGANATAEAISLTQRFNDSGIVGCLTVTPYYNRPSQEGLYQHFKAIAEHTDLPQILYNVPSRTGCDLLPETVGRLAKVKNIIGIKEATGNLTRVNQIKELVSDDFVLLSGDDASALDFMQLGGHGVISVTANVAARDMAQMCKLAAEGHFAEARVINQRLMPLHNKLFVEPNPIPVKWACKELGLVATDTLRLPMTPITDSGRETVRAALKHAGLLSEQ ID NO: 5 (Escherichia coli meso-diaminopimelate decarboxylasepolypeptide sequence, LysA, encoded by lysA gene)MPHSLFSTDTDLTAENLLRLPAEFGCPVWVYDAQIIRRQIAALKQFDVVRFAQKACSNIHILRLMREQGVKVDSVSLGEIERALAAGYNPQTHPDDIVFTADVIDQATLERVSELQIPVNAGSVDMLDQLGQVSPGHRVWLRVNPGFGHGHSQKTNTGGENSKHGIWYTDLPAALDVIQRHHLQLVGIHMHIGSGVDYAHLEQVCGAMVRQVIEFGQDLQAISAGGGLSVPYQQGEEAVDTEHYYGLWNAAREQIARHLGHPVKLEIEPGRFLVAQSGVLITQVRSVKQMGSRHFVLVDAGFNDLMRPAMYGSYHHISALAADGRSLEHAPTVETVVAGPLCESGDVFTQQEGGNVETRALPEVKAGDYLVLHDTGAYGASMSSNYNSRPLLPEVLFDNGQARLIRRRQTIEELLALELLSEQ ID NO: 6 (Escherichia coli CadA polypeptide sequence, encodedby cadA gene) MNVIAILNHMGVYFKEEPIRELHRALERLNFQIVYPNDRDDLLKLIENNARLCGVIFDWDKYNLELCEEISKMNENLPLYAFANTYSTLDVSLNDLRLQISFFEYALGAAEDIANKIKQTTDEYINTILPPLTKALFKYVREGKYTFCTPGHMGGTAFQKSPVGSLFYDFFGPNTMKSDISISVSELGSLLDHSGPHKEAEQYIARVFNADRSYMVTNGTSTANKIVGMYSAPAGSTILIDRNCHKSLTHLMMMSDVTPIYFRPTRNAYGILGGIPQSEFQHATIAKRVKETPNATWPVHAVITNSTYDGLLYNTDFIKKTLDVKSIHFDSAWVPYTNFSPIYEGKCGMSGGRVEGKVIYETQSTHKLLAAFSQASMIHVKGDVNEETFNEAYMMHTTTSPHYGIVASTETAAAMMKGNAGKRLINGSIERAIKFRKEIKRLRTESDGWFFDVWQPDHIDTTECWPLRSDSTWHGFKNIDNEHMYLDPIKVTLLTPGMEKDGTMSDFGIPASIVAKYLDEHGIVVEKTGPYNLLFLFSIGIDKTKALSLLRALTDFKRAFDLNLRVKNMLPSLYREDPEFYENMRIQELAQNIHKLIVHHNLPDLMYRAFEVLPTMVMTPYAAFQKELHGMTEEVYLDEMVGRINANMILPYPPGVPLVMPGEMITEESRPVLEFLQMLCEIGAHYPGFETDIHGAYRQADGRYTVKVLKEESKKSEQ ID NO: 7 (Escherichia coli LdcC polypeptide sequence (encodedby ldcC gene))MNIIAIMGPHGVFYKDEPIKELESALVAQGFQIIWPQNSVDLLKFIEHNPRICGVIFDWDEYSLDLCSDINQLNEYLPLYAFINTHSTMDVSVQDMRMALWFFEYALGQAEDIAIRMRQYTDEYLDNITPPFTKALFTYVKERKYTFCTPGHMGGTAYQKSPVGCLFYDFFGGNTLKADVSISVTELGSLLDHTGPHLEAEEYIARTFGAEQSYIVTNGTSTSNKIVGMYAAPSGSTLLIDRNCHKSLAHLLMMNDVVPVWLKPTRNALGILGGIPRREFTRDSIEEKVAATTQAQWPVHAVITNSTYDGLLYNTDWIKQTLDVPSIHFDSAWVPYTHFHPIYQGKSGMSGERVAGKVIFETQSTHKMLAALSQASLIHIKGEYDEEAFNEAFMMHTTTSPSYPIVASVETAAAMLRGNPGKRLINRSVERALHFRKEVQRLREESDGWFFDIWQPPQVDEAECWPVAPGEQWHGFNDADADHMFLDPVKVTILTPGMDEQGNMSEEGIPAALVAKFLDERGIVVEKTGPYNLLFLFSIGIDKTKAMGLLRGLTEFKRSYDLNLRIKNMLPDLYAEDPDFYRNMRIQDLAQGIHKLIRKHDLPGLMLRAFDTLPEMIMTPHQAWQRQIKGEVETIALEQLVGRVSANMILPYPPGVPLLMPGEMLTKESRTVLDFLLMLCSVGQHYPGFETDIHGAKQDEDGVYR VRVLKMAGSEQ ID NO: 8 (Pseudomonas aeruginosa ldc2 polynucleotide  sequence) ATGTATAAAGACCTCAAATTTCCCGTCCTCATCGTCCATCGCGACATCAAGGCCGACACCGTTGCCGGCGAACGCGTGCGGGGCATCGCCCACGAACTGGAGCAGGACGGCTTCAGCATTCTCTCCACCGCCAGCTCCGCCGAGGGGCGCATCGTCGCTTCCACCCACCACGGCCTGGCCTGCATTCTGGTCGCCGCCGAAGGTGCCGGGGAAAACCAGCGCCTGCTGCAGGATGTGGTCGAACTGATCCGCGTGGCCCGCGTGCGGGCGCCGCAATTGCCGATCTTCGCCCTCGGCGAGCAGGTGACCATCGAGAACGCGCCGGCCGAGTCCATGGCCGACCTGCACCAGTTGCGCGGCATCCTCTACCTGTTCGAAGACACCGTGCCGTTCCTCGCCCGCCAGGTCGCCCGGGCGGCGCGCAACTACCTGGCCGGGCTGCTGCCGCCATTCTTCCGTGCGCTGGTCGAGCACACCGCGCAGTCCAACTATTCCTGGCATACGCCGGGCCACGGCGGCGGTGTCGCCTATCGCAAGAGTCCGGTGGGACAGGCGTTCCACCAGTTCTTCGGGGAGAACACGCTGCGTTCCGACCTGTCGGTCTCGGTCCCCGAGCTGGGATCGCTGCTCGACCATACCGGCCCCCTGGCCGAGGCCGAGGACCGTGCCGCGCGCAATTTCGGCGCCGACCATACCTTCTTCGTGATCAATGGCACTTCCACCGCGAACAAGATCGTCTGGCACTCCATGGTCGGTCGCGAAGACCTGGTGCTGGTGGACCGCAACTGCCACAAGTCGATCCTCCACTCGATCATCATGACCGGGGCGATACCGCTCTACCTGACTCCGGAACGCAACGAACTGGGGATCATCGGGCCGATCCCGCTGAGCGAATTCAGCAAGCAGTCGATCGCCGCGAAGATCGCCGCCAGCCCGCTGGCGCGCGGCCGCGAGCCGAAGGTGAAGCTGGCGGTGGTGACTAACTCCACCTACGACGGCCTGTGCTACAACGCCGAGCTGATCAAGCAGACCCTCGGCGACAGCGTCGAGGTGTTGCACTTCGACGAGGCTTGGTACGCCTATGCCGCGTTCCACGAGTTCTACGACGGACGCTATGGCATGGGCACCTCGCGCAGCGAGGAGGGACCCCTGGTGTTCGCCACCCACTCCACGCACAAGATGCTCGCCGCCTTCAGCCAGGCCTCGATGATCCACGTGCAGGATGGCGGGACCCGGAAGCTGGACGTGGCGCGCTTCAACGAAGCCTTCATGATGCACATCTCGACCTCGCCGCAGTACGGCATCATCGCTTCGCTGGACGTGGCTTCGGCGATGATGGAAGGGCCCGCCGGGCGTTCGCTGATCCAGGAGACCTTCGACGAGGCCCTCAGCTTCCGCCGGGCCCTGGCCAACGTACGGCAGAACCTGGACCGGAACGACTGGTGGTTCGGCGTCTGGCAGCCGGAGCAGGTGGAGGGCACCGACCAGGTCGGCACCCATGACTGGGTGCTGGAGCCGAGCGCCGACTGGCACGGCTTCGGCGATATCGCCGAAGACTACGTGCTGCTCGACCCGATCAAGGTCACCCTGACCACCCCGGGCCTGAGCGCTGGCGGCAAGCTCAGCGAGCAGGGGATTCCGGCCGCCATCGTCAGCCGCTTCCTCTGGGAGCGCGGGCTGGTGGTGGAGAAAACCGGTCTCTACTCCTTCCTGGTGCTGTTCTCGATGGGCATCACCAAGGGCAAGTGGAGCACCCTGGTCACCGAACTGCTCGAATTCAAGCGCTGTTACGACGCCAACCTGCCGCTGCTTGACGTCTTGCCCTCCGTGGCCCAGGCCGGCGGCAAGCGCTACAACGGAGTGGGCCTGCGCGACCTCAGCGACGCCATGCACGCCAGCTACCGCGACAACGCCACGGCGAAGGCCATGAAGCGCATGTACACGGTGCTGCCGGAGGTCGCGATGCGGCCGTCCGAGGCCTACGACAAGCTGGTGCGCGGCGAGGTCGAGGCGGTACCGATCGCTCGGTTGGAAGGGCGCATCGCGGCCGTCATGCTGGTACCCTATCCGCCGGGTATCCCGCTGATCATGCCGGGTGAGCGCTTCACCGAGGCGACCCGCTCGATCCTCGACTATCTCGAGTTCGCGCGGACCTTCGAGCGCGCCTTCCCTGGTTTCGACTCCGATGTGCATGGCCTGCAGCATCAGGACGGACCGTCCGGGCGCTGCTATACCGTTGAATGCATAAAGGAATGASEQ ID NO: 9 (Pseudomonas aeruginosa Ldc2 polypeptide sequence (encoded by ldc2 gene)) MYKDLKFPVLIVHRDIKADTVAGERVRGIAHELEQDGFSILSTASSAEGRIVASTHHGLACILVAAEGAGENQRLLQDVVELIRVARVRAPQLPIFALGEQVTIENAPAESMADLHQLRGILYLFEDTVPFLARQVARAARNYLAGLLPPFFRALVEHTAQSNYSWHTPGHGGGVAYRKSPVGQAFHQFFGENTLRSDLSVSVPELGSLLDHTGPLAEAEDRAARNFGADHTFFVINGTSTANKIVWHSMVGREDLVLVDRNCHKSILHSIIMTGAIPLYLTPERNELGIIGPIPLSEFSKQSIAAKIAASPLARGREPKVKLAVVTNSTYDGLCYNAELIKQTLGDSVEVLHFDEAWYAYAAFHEFYDGRYGMGTSRSEEGPLVFATHSTHKMLAAFSQASMIHVQDGGTRKLDVARFNEAFMMHISTSPQYGIIASLDVASAMMEGPAGRSLIQETFDEALSFRRALANVRQNLDRNDWWFGVWQPEQVEGTDQVGTHDWVLEPSADWHGFGDIAEDYVLLDPIKVTLTTPGLSAGGKLSEQGIPAAIVSRFLWERGLVVEKTGLYSFLVLFSMGITKGKWSTLVTELLEFKRCYDANLPLLDVLPSVAQAGGKRYNGVGLRDLSDAMHASYRDNATAKAMKRMYTVLPEVAMRPSEAYDKLVRGEVEAVPIARLEGRIAAVMLVPYPPGIPLIMPGERFTEATRSILDYLEFARTFERAFPGFDSDVHGLQHQDGPSGRCYTVECIKESEQ ID NO: 10 (Pseudomonas aeruginosa (ldc2-co1 DNA sequence)) ATGTACAAAGATCTGAAATTCCCTGTTCTGATTGTACACCGCGATATCAAGGCGGACACGGTAGCCGGCGAGCGTGTTCGCGGTATTGCCCACGAACTCGAACAAGACGGCTTTAGCATTCTCTCTACGGCGTCTTCTGCGGAAGGCCGCATTGTGGCTAGCACGCACCACGGTCTCGCCTGCATCCTCGTGGCAGCTGAGGGTGCGGGTGAGAATCAGCGTCTGCTCCAAGACGTGGTTGAGCTGATCCGTGTAGCTCGCGTCCGTGCGCCACAGCTCCCGATCTTCGCGCTGGGCGAACAGGTGACTATTGAAAACGCGCCTGCCGAATCTATGGCCGACCTGCACCAGCTCCGCGGCATTCTGTATCTCTTCGAGGATACTGTCCCGTTCCTGGCACGTCAGGTTGCACGCGCAGCGCGTAACTACCTCGCTGGCCTCCTCCCGCCATTCTTCCGTGCACTCGTGGAGCACACGGCCCAAAGCAATTACTCTTGGCACACCCCGGGTCACGGTGGTGGTGTCGCTTACCGTAAATCTCCGGTAGGTCAAGCTTTCCACCAGTTCTTTGGCGAGAATACCCTCCGCTCTGACCTGTCTGTTAGCGTTCCAGAGCTGGGCAGCCTGCTGGATCACACTGGCCCTCTCGCGGAAGCAGAGGATCGTGCCGCTCGCAATTTCGGTGCGGACCACACCTTCTTTGTCATCAATGGTACCTCTACTGCGAACAAAATCGTTTGGCACTCTATGGTTGGTCGCGAGGACCTGGTGCTGGTCGATCGTAACTGTCACAAATCTATTCTGCACTCCATTATCATGACGGGTGCTATCCCACTGTACCTGACTCCGGAACGCAACGAACTGGGTATTATCGGCCCTATTCCACTCTCCGAGTTTTCTAAACAATCTATCGCAGCAAAAATTGCCGCCTCCCCACTCGCGCGTGGTCGTGAACCGAAAGTTAAACTGGCTGTCGTTACCAACTCTACCTATGACGGTCTGTGTTACAACGCGGAACTGATCAAACAAACCCTCGGCGACTCTGTCGAGGTACTGCATTTCGACGAGGCTTGGTATGCTTATGCGGCGTTTCACGAGTTCTACGACGGCCGCTACGGTATGGGCACTTCTCGTTCCGAAGAGGGTCCGCTGGTCTTTGCTACCCATTCTACCCACAAGATGCTCGCGGCTTTTTCCCAAGCTAGCATGATTCACGTTCAGGATGGTGGTACGCGCAAGCTGGACGTCGCCCGCTTTAACGAAGCCTTTATGATGCACATCAGCACCTCTCCACAGTACGGCATCATTGCGTCTCTCGATGTCGCAAGCGCTATGATGGAAGGTCCTGCCGGTCGTAGCCTGATCCAAGAGACGTTCGATGAGGCGCTGTCCTTCCGTCGTGCTCTGGCGAATGTCCGTCAGAACCTGGACCGTAATGATTGGTGGTTCGGTGTCTGGCAACCGGAGCAGGTTGAGGGCACCGACCAGGTAGGTACTCACGACTGGGTTCTCGAGCCTAGCGCGGACTGGCATGGTTTTGGTGACATTGCGGAGGATTACGTTCTCCTCGATCCTATCAAAGTTACCCTGACCACCCCAGGTCTGAGCGCTGGCGGTAAACTCTCTGAACAAGGCATCCCGGCAGCTATCGTTAGCCGTTTCCTGTGGGAACGTGGTCTGGTGGTCGAGAAAACGGGTCTGTACTCTTTCCTGGTTCTGTTCTCCATGGGTATCACGAAAGGCAAATGGTCTACTCTGGTTACCGAGCTGCTCGAATTCAAACGCTGTTACGACGCGAATCTGCCACTCCTGGATGTGCTGCCTTCTGTAGCGCAGGCGGGTGGTAAACGCTATAACGGTGTAGGTCTGCGTGATCTGTCCGATGCCATGCACGCTTCTTATCGTGACAATGCCACGGCGAAGGCCATGAAGCGTATGTATACGGTGCTCCCGGAAGTAGCCATGCGCCCGTCCGAAGCTTATGATAAGCTCGTACGCGGTGAAGTCGAAGCTGTTCCTATTGCACGTCTCGAGGGTCGTATTGCGGCGGTTATGCTGGTTCCGTACCCGCCAGGTATCCCGCTCATTATGCCGGGTGAACGTTTTACTGAAGCTACCCGCTCCATTCTGGACTATCTGGAGTTTGCCCGTACCTTCGAGCGCGCGTTCCCGGGCTTTGACTCTGATGTTCACGGCCTCCAACATCAAGATGGCCCGTCTGGCCGTTGTTATACCGTTGAATGCATCAAGGAATAASEQ ID NO: 11 (Pseudomonas aeruginosa mutant Ldc2 S111Cpolypeptide sequence (encoded by mutant ldc2 gene))MYKDLKFPVLIVHRDIKADTVAGERVRGIAHELEQDGFSILSTASSAEGRIVASTHHGLACILVAAEGAGENQRLLQDVVELIRVARVRAPQLPIFALGEQVTIENAPAECMADLHQLRGILYLFEDTVPFLARQVARAARNYLAGLLPPFFRALVEHTAQSNYSWHTPGHGGGVAYRKSPVGQAFHQFFGENTLRSDLSVSVPELGSLLDHTGPLAEAEDRAARNFGADHTFFVINGTSTANKIVWHSMVGREDLVLVDRNCHKSILHSIIMTGAIPLYLTPERNELGIIGPIPLSEFSKQSIAAKIAASPLARGREPKVKLAVVTNSTYDGLCYNAELIKQTLGDSVEVLHFDEAWYAYAAFHEFYDGRYGMGTSRSEEGPLVFATHSTHKMLAAFSQASMIHVQDGGTRKLDVARFNEAFMMHISTSPQYGIIASLDVASAMMEGPAGRSLIQETFDEALSFRRALANVRQNLDRNDWWFGVWQPEQVEGTDQVGTHDWVLEPSADWHGFGDIAEDYVLLDPIKVTLTTPGLSAGGKLSEQGIPAAIVSRFLWERGLVVEKTGLYSFLVLFSMGITKGKWSTLVTELLEFKRCYDANLPLLDVLPSVAQAGGKRYNGVGLRDLSDAMHASYRDNATAKAMKRMYTVLPEVAMRPSEAYDKLVRGEVEAVPIARLEGRIAAVMLVPYPPGIPLIMPGERFTEATRSILDYLEFARTFERAFPGFDSDVHGLQHQDGPSGRCYTVECIKE SEQ ID NO: 12 (aat) >gb|AY271828.1|:385-1717 H. alvei plasmid pAlvA, complete sequence    1ttgactttgt taaaagtcag gcataagatc aaaatactgt atatataaca atgtatttat   61atacagtatt ttatactttt tatctaacgt cagagagggc aatattatga gtggtggaga  121tggcaagggt cacaatagtg gagcacatga ttccggtggc agcattaatg gaacttctgg  181gaaaggtggg ccatcaagcg gaggagcatc agataattct gggtggagtt cggaaaataa  241cccgtggggc ggtggtaact cgggaatgat tggtggcagt caaggaggta acggagctaa  301tcatggtggc gaaaatacat cttctaacta tgggaaagat gtatcacgcc aaatcggtga  361tgcgatagcc agaaaggaag gcatcaatcc gaaaatattc actgggtact ttatccgttc  421agatggatat ttgatcggaa taacgccact tgtcagtggt gatgcctttg gcgttaatct  481tggcctgttc aataacaatc aaaatagtag tagtgaaaat aagggatgga atggaaggaa  541tggagatggc attaaaaata gtagccaagg tggatggaag attaaaacta atgaacttac  601ttcaaaccaa gtagctgctg ctaaatccgt tccagaacct aaaaatagta aatattataa  661gtccatgaga gaagctagcg atgaggttat taattctaat ttaaaccaag ggcatggagt  721tggtgaggca gctagagctg aaagagatta cagagaaaaa gtaaagaacg caatcaatga  781taatagtccc aatgtgctac aggatgctat taaatttaca gcagattttt ataaggaagt  841ttttaacgct tacggagaaa aagccgaaaa actagccaag ttattagctg atcaagctaa  901aggtaaaaag atccgcaatg tagaagatgc attgaaatct tatgaaaaac acaaggctaa  961cattaacaaa aaaatcaatg cgaaagatcg cgaagctatc gccaaggctt tggagtctat 1021ggatgtagaa aaagccgcaa aaaatatatc caagttcagc aaaggactag gttgggttgg 1081cccagctatc gatataactg attggtttac agaattatac aaagcagtga aaactgataa 1141ttggagatct ctttatgtta aaactgaaac tattgcagta gggctagctg caacccatgt 1201caccgcctta gcattcagtg ctgtcttggg tgggcctata ggtattttag gttatggttt 1261gattatggct ggggttgggg cgttagttaa cgagacaata gttgacgagg caaataaggt 1321cattgggatt taa SEQ ID NO: 13 (aai) >gb|AY271828.1|:1734-2069 HI alvei plasmid pAlvA, complete sequence   1ctatatttta gcggtcacat tttttatttc aaaacaaaca gaaagaacac caataggaat  61tgatgtcata aaaataaaaa taaaatacaa agtcattaaa tatgtttttg gcacaccatc 121cttaaaaaaa cctgttttcc aaaattcttt tttcgtatat ctaagcgctg ctttctctat 181tagaaaccga gagaaaggaa atagaatagc gctagccaaa ccaaagattc tgagcgcaat 241tattttaggt tcgtcatcac cataactggc gtaaagaata caagcagcca taaagtatcc 301ccaaaacata ttatgtatgt aatatttcct tgtcat SEQ ID NO: 14 (abt) >gb|AY271829.1|:384-1566 H. alvei plasmid pAlvB, complete sequence    1atgagtggtg gagacggtaa aggtcacaat agtggagcac atgattccgg tggcagcatt   61aatggaactt cggggaaagg tggacctgat tctggtggcg gatattggga caaccatcca  121catattacaa tcaccggtgg acgggaagta ggtcaagggg gagctggtat caactggggt  181ggtggttctg gtcatggtaa cggcgggggc tcagttgcca tccaagaata taacacgagt  241aaatatccta acacgggagg atttcctcct cttggagacg ctagctggct gttaaatcct  301ccaaaatggt cggttattga agtaaaatca gaaaactcag catggcgctc ttatattact  361catgttcaag gtcatgttta caaattgact tttgatggta cgggtaagct cattgatacc  421gcgtatgtta attatgaacc cagtgatgat actcgttgga gcccgcttaa aagttttaaa  481tataataaag gaaccgctga aaaacaggtt agggatgcca ttaacaatga aaaagaagca  541gttaaggacg ctgttaaatt tactgcagac ttctataaag aggtttttaa ggtttacgga  601gaaaaagccg agaagctcgc taagttatta gcagatcaag ctaaaggcaa aaaggttcgc  661aacgtagaag atgccttgaa atcttatgaa aaatataaga ctaacattaa caaaaaaatc  721aatgcgaaag atcgcgaagc tattgctaaa gccttggagt ctatggatgt aggaaaagcc  781gcaaaaaata tagccaagtt cagtaaagga ctaggttggg ttggccctgc tatcgatata  841actgattggt ttacagaatt atacaaggca gtggaaactg ataattggag atctttttat  901gttaaaactg aaactattgc agtagggcta gctgcaaccc atgttgccgc cttggcattc  961agcgctgtct tgggtgggcc tgtaggtatt ttgggttatg gtttgattat ggctggggtt 1021ggggcgttag ttaatgagac aatagttgac gaggcaaata aggttattgg gctttaaSEQ ID NO: 15 (abi) >gb|AY271829.1|:1583-1918 H. alvei plasmid pAlvB, complete sequence    1ctataattta gcggtcacat tttttatttc aaaaaaaaca gaaataacac ctataggaat   61tgatgtcata aaaataaaaa ttaaatacaa agtcattaaa tatgtttttg gcacgccatc  121cttaaaaaaa ccagtttccc aaaattcttt tttcgtatat ctaagcgcgg ttttctctat  181taaaaaccga gagaaaggga ataggatagc actagccaaa ccaaagattc tgagcgcaat  241tattttaggt tcgttatccc cataactggc gtaaagaata caaacagcca taaagtaccc  301ccaaaacata ttatgtatat aatatttcct tgtcat SEQ ID NO: 16 (Ldc2 N262T protein sequence)MYKDLKFPVLIVHRDIKADTVAGERVRGIAHELEQDGFSILSTASSAEGRIVASTHHGLACILVAAEGAGENQRLLQDVVELIRVARVRAPQLPIFALGEQVTIENAPAESMADLHQLRGILYLFEDTVPFLARQVARAARNYLAGLLPPFFRALVEHTAQSNYSWHTPGHGGGVAYRKSPVGQAFHQFFGENTLRSDLSVSVPELGSLLDHTGPLAEAEDRAARNFGADHTFFVINGTSTANKIVWHSMVGREDLVLVDRTCHKSILHSIIMTGAIPLYLTPERNELGIIGPIPLSEFSKQSIAAKIAASPLARGREPKVKLAVVTNSTYDGLCYNAELIKQTLGDSVEVLHFDEAWYAYAAFHEFYDGRYGMGTSRSEEGPLVFATHSTHKMLAAFSQASMIHVQDGGTRKLDVARFNEAFMMHISTSPQYGIIASLDVASAMMEGPAGRSLIQETFDEALSFRRALANVRQNLDRNDWWFGVWQPEQVEGTDQVGTHDWVLEPSADWHGFGDIAEDYVLLDPIKVTLTTPGLSAGGKLSEQGIPAAIVSRFLWERGLVVEKTGLYSFLVLFSMGITKGKWSTLVTELLEFKRCYDANLPLLDVLPSVAQAGGKRYNGVGLRDLSDAMHASYRDNATAKAMKRMYTVLPEVAMRPSEAYDKLVRGEVEAVPIARLEGRIAAVMLVPYPPGIPLIMPGERFTEATRSILDYLEFARTFERAFPGFDSDVHGLQHQDGPSGRCYTVECIKESEQ ID NO: 17 (Ldc2 K265N protein sequence)MYKDLKFPVLIVHRDIKADTVAGERVRGIAHELEQDGFSILSTASSAEGRIVASTHHGLACILVAAEGAGENQRLLQDVVELIRVARVRAPQLPIFALGEQVTIENAPAESMADLHQLRGILYLFEDTVPFLARQVARAARNYLAGLLPPFFRALVEHTAQSNYSWHTPGHGGGVAYRKSPVGQAFHQFFGENTLRSDLSVSVPELGSLLDHTGPLAEAEDRAARNFGADHTFFVINGTSTANKIVWHSMVGREDLVLVDRNCHNSILHSIIMTGAIPLYLTPERNELGIIGPIPLSEFSKQSIAAKIAASPLARGREPKVKLAVVTNSTYDGLCYNAELIKQTLGDSVEVLHFDEAWYAYAAFHEFYDGRYGMGTSRSEEGPLVFATHSTHKMLAAFSQASMIHVQDGGTRKLDVARFNEAFMMHISTSPQYGIIASLDVASAMMEGPAGRSLIQETFDEALSFRRALANVRQNLDRNDWWFGVWQPEQVEGTDQVGTHDWVLEPSADWHGFGDIAEDYVLLDPIKVTLTTPGLSAGGKLSEQGIPAAIVSRFLWERGLVVEKTGLYSFLVLFSMGITKGKWSTLVTELLEFKRCYDANLPLLDVLPSVAQAGGKRYNGVGLRDLSDAMHASYRDNATAKAMKRMYTVLPEVAMRPSEAYDKLVRGEVEAVPIARLEGRIAAVMLVPYPPGIPLIMPGERFTEATRSILDYLEFARTFERAFPGFDSDVHGLQHQDGPSGRCYTVECIKESEQ ID NO: 18 (Ldc2 S111C/N262T protein sequence)MYKDLKFPVLIVHRDIKADTVAGERVRGIAHELEQDGFSILSTASSAEGRIVASTHHGLACILVAAEGAGENQRLLQDVVELIRVARVRAPQLPIFALGEQVTIENAPAECMADLHQLRGILYLFEDTVPFLARQVARAARNYLAGLLPPFFRALVEHTAQSNYSWHTPGHGGGVAYRKSPVGQAFHQFFGENTLRSDLSVSVPELGSLLDHTGPLAEAEDRAARNFGADHTFFVINGTSTANKIVWHSMVGREDLVLVDRTCHKSILHSIIMTGAIPLYLTPERNELGIIGPIPLSEFSKQSIAAKIAASPLARGREPKVKLAVVTNSTYDGLCYNAELIKQTLGDSVEVLHFDEAWYAYAAFHEFYDGRYGMGTSRSEEGPLVFATHSTHKMLAAFSQASMIHVQDGGTRKLDVARFNEAFMMHISTSPQYGIIASLDVASAMMEGPAGRSLIQETFDEALSFRRALANVRQNLDRNDWWFGVWQPEQVEGTDQVGTHDWVLEPSADWHGFGDIAEDYVLLDPIKVTLTTPGLSAGGKLSEQGIPAAIVSRFLWERGLVVEKTGLYSFLVLFSMGITKGKWSTLVTELLEFKRCYDANLPLLDVLPSVAQAGGKRYNGVGLRDLSDAMHASYRDNATAKAMKRMYTVLPEVAMRPSEAYDKLVRGEVEAVPIARLEGRIAAVMLVPYPPGIPLIMPGERFTEATRSILDYLEFARTFERAFPGFDSDVHGLQHQDGPSGRCYTVECIKESEQ ID NO: 19 (Ldc2 S111C/K265N protein sequence)MYKDLKFPVLIVHRDIKADTVAGERVRGIAHELEQDGFSILSTASSAEGRIVASTHHGLACILVAAEGAGENQRLLQDVVELIRVARVRAPQLPIFALGEQVTIENAPAECMADLHQLRGILYLFEDTVPFLARQVARAARNYLAGLLPPFFRALVEHTAQSNYSWHTPGHGGGVAYRKSPVGQAFHQFFGENTLRSDLSVSVPELGSLLDHTGPLAEAEDRAARNFGADHTFFVINGTSTANKIVWHSMVGREDLVLVDRNCHNSILHSIIMTGAIPLYLTPERNELGIIGPIPLSEFSKQSIAAKIAASPLARGREPKVKLAVVTNSTYDGLCYNAELIKQTLGDSVEVLHFDEAWYAYAAFHEFYDGRYGMGTSRSEEGPLVFATHSTHKMLAAFSQASMIHVQDGGTRKLDVARFNEAFMMHISTSPQYGIIASLDVASAMMEGPAGRSLIQETFDEALSFRRALANVRQNLDRNDWWFGVWQPEQVEGTDQVGTHDWVLEPSADWHGFGDIAEDYVLLDPIKVTLTTPGLSAGGKLSEQGIPAAIVSRFLWERGLVVEKTGLYSFLVLFSMGITKGKWSTLVTELLEFKRCYDANLPLLDVLPSVAQAGGKRYNGVGLRDLSDAMHASYRDNATAKAMKRMYTVLPEVAMRPSEAYDKLVRGEVEAVPIARLEGRIAAVMLVPYPPGIPLIMPGERFTEATRSILDYLEFARTFERAFPGFDSDVHGLQHQDGPSGRCYTVECIKESEQ ID NO: 20 (Ldc2 N262T/K265N protein sequence)MYKDLKFPVLIVHRDIKADTVAGERVRGIAHELEQDGFSILSTASSAEGRIVASTHHGLACILVAAEGAGENQRLLQDVVELIRVARVRAPQLPIFALGEQVTIENAPAESMADLHQLRGILYLFEDTVPFLARQVARAARNYLAGLLPPFFRALVEHTAQSNYSWHTPGHGGGVAYRKSPVGQAFHQFFGENTLRSDLSVSVPELGSLLDHTGPLAEAEDRAARNFGADHTFFVINGTSTANKIVWHSMVGREDLVLVDRTCHNSILHSIIMTGAIPLYLTPERNELGIIGPIPLSEFSKQSIAAKIAASPLARGREPKVKLAVVTNSTYDGLCYNAELIKQTLGDSVEVLHFDEAWYAYAAFHEFYDGRYGMGTSRSEEGPLVFATHSTHKMLAAFSQASMIHVQDGGTRKLDVARFNEAFMMHISTSPQYGIIASLDVASAMMEGPAGRSLIQETFDEALSFRRALANVRQNLDRNDWWFGVWQPEQVEGTDQVGTHDWVLEPSADWHGFGDIAEDYVLLDPIKVTLTTPGLSAGGKLSEQGIPAAIVSRFLWERGLVVEKTGLYSFLVLFSMGITKGKWSTLVTELLEFKRCYDANLPLLDVLPSVAQAGGKRYNGVGLRDLSDAMHASYRDNATAKAMKRMYTVLPEVAMRPSEAYDKLVRGEVEAVPIARLEGRIAAVMLVPYPPGIPLIMPGERFTEATRSILDYLEFARTFERAFPGFDSDVHGLQHQDGPSGRCYTVECIKESEQ ID NO: 21 (Ldc2 S111C/N262T/K265N protein sequence)MYKDLKFPVLIVHRDIKADTVAGERVRGIAHELEQDGFSILSTASSAEGRIVASTHHGLACILVAAEGAGENQRLLQDVVELIRVARVRAPQLPIFALGEQVTIENAPAECMADLHQLRGILYLFEDTVPFLARQVARAARNYLAGLLPPFFRALVEHTAQSNYSWHTPGHGGGVAYRKSPVGQAFHQFFGENTLRSDLSVSVPELGSLLDHTGPLAEAEDRAARNFGADHTFFVINGTSTANKIVWHSMVGREDLVLVDRTCHNSILHSIIMTGAIPLYLTPERNELGIIGPIPLSEFSKQSIAAKIAASPLARGREPKVKLAVVTNSTYDGLCYNAELIKQTLGDSVEVLHFDEAWYAYAAFHEFYDGRYGMGTSRSEEGPLVFATHSTHKMLAAFSQASMIHVQDGGTRKLDVARFNEAFMMHISTSPQYGIIASLDVASAMMEGPAGRSLIQETFDEALSFRRALANVRQNLDRNDWWFGVWQPEQVEGTDQVGTHDWVLEPSADWHGFGDIAEDYVLLDPIKVTLTTPGLSAGGKLSEQGIPAAIVSRFLWERGLVVEKTGLYSFLVLFSMGITKGKWSTLVTELLEFKRCYDANLPLLDVLPSVAQAGGKRYNGVGLRDLSDAMHASYRDNATAKAMKRMYTVLPEVAMRPSEAYDKLVRGEVEAVPIARLEGRIAAVMLVPYPPGIPLIMPGERFTEATRSILDYLEFARTFERAFPGFDSDVHGLQHQDGPSGRCYTVECIKESEQ ID NO: 22 (Escherichia coli Asd polypeptide sequence (encodedby asd gene))  MKNVGFIGWRGMVGSVLMQRMVEERDFDAIRPVFFSTSQLGQAAPSFGGTTGTLQDAFDLEALKALDIIVTCQGGDYTNEIYPKLRESGWQGYWIDAASSLRMKDDAIIILDPVNQDVITDGLNNGIRTFVGGNCTVSLMLMSLGGLFANDLVDWVSVATYQAASGGGARHMRELLTQMGHLYGHVADELATPSSAILDIERKVTTLTRSGELPVDNFGVPLAGSLIPWIDKQLDNGQSREEWKGQAETNKILNTSSVIPVDGLCVRVGALRCHSQAFTIKLKKDVSIPTVEELLAAHNPWAKVVPNDREITMRELTPAAVTGTLTTPVGRLRKLNMGPEFLSAFTVGDQ LLWGAAEPLRRMLRQLASEQ ID NO: 23 (Escherichia coli DapB polypeptide sequence(encoded by dapB gene))MHDANIRVAIAGAGGRMGRQLIQAALALEGVQLGAALEREGSSLLGSDAGELAGAGKTGVTVQSSLDAVKDDFDVFIDFTRPEGTLNHLAFCRQHGKGMVIGTTGFDEAGKQAIRDAAADIAIVFAANFSVGVNVMLKLLEKAAKVMGDYTDIFIIEAHHRHKVDAPSGTALAMGEAIAHALDKDLKDCAVYSREGHTGERVPGTIGFATVRAGDIVGEHTAMFADIGERLEITHKASSRMTFANGAVRSALWLSGKESGLFDMRDVLDLNNLSEQ ID NO: 24 (Escherichia coli DapD polypeptide sequence(encoded by dapD gene))MQQLQNIIETAFERRAEITPANADTVTREAVNQVIALLDSGALRVAEKIDGQWVTHQWLKKAVLLSFRINDNQVIEGAESRYFDKVPMKFADYDEARFQKEGFRVVPPAAVRQGAFIARNTVLMPSYVNIGAYVDEGTMVDTWATVGSCAQIGKNVHLSGGVGIGGVLEPLQANPTIIEDNCFIGARSEVVEGVIVEEGSVISMGVYIGQSTRIYDRETGEIHYGRVPAGSVVVSGNLPSKDGKYSLYCAVIVKKVDAKTRGKVGINELLRTIDSEQ ID NO: 25 (Escherichia coli AspC polypeptide sequence(encoded by aspC gene))MFENITAAPADPILGLADLFRADERPGKINLGIGVYKDETGKTPVLTSVKKAEQYLLENETTKNYLGIDGIPEFGRCTQELLFGKGSALINDKRARTAQTPGGTGALRVAADFLAKNTSVKRVWVSNPSWPNHKSVFNSAGLEVREYAYYDAENHTLDFDALINSLNEAQAGDVVLFHGCCHNPTGIDPTLEQWQTLAQLSVEKGWLPLFDFAYQGFARGLEEDAEGLRAFAAMHKELIVASSYSKNFGLYNERVGACTLVAADSETVDRAFSQMKAAIRANYSNPPAHGASVVATILSNDALRAIWEQELTDMRQRIQRMRQLFVNTLQEKGANRDFSFIIKQNGMFSFSGLTKEQVLRLREEFGVYAVASGRVNVAGMTPDNMAPLCEAIVAVLSEQ ID NO: 26 (Escherichia coli mutant aspartokinase III polypeptidesequence, LysC-1 (M318I, G323D))MSEIVVSKFGGTSVADFDAMNRSADIVLSDANVRLVVLSASAGITNLLVALAEGLEPGERFEKLDAIRNIQFAILERLRYPNVIREEIERLLENITVLAEAAALATSPALTDELVSHGELMSTLLFVEILRERDVQAQWFDVRKVMRTNDRFGRAEPDIAALAELAALQLLPRLNEGLVITQGFIGSENKGRTTTLGRGGSDYTAALLAEALHASRVDIWTDVPGIYTTDPRVVSAAKRIDEIAFAEAAEMATFGAKVLHPATLLPAVRSDIPVFVGSSKDPRAGGTLVCNKTENPPLFRALALRRNQTLLTLHSLNILHSRDFLAEVFGILARHNISVDLITTSEVSVALTLDTTGSTSTGDTLLTQSLLMELSALCRVEVEEGLALVALIGNDLSKACGVGKEVFGVLEPFNIRMICYGASSHNLCFLVPGEDAEQVVQKLHSNLFESEQ ID NO: 27 (Escherichia coli mutant aspartokinase III polypeptidesequence, LysC-2 (T344M, T352I)) MSEIVVSKFGGTSVADFDAMNRSADIVLSDANVRLVVLSASAGITNLLVALAEGLEPGERFEKLDAIRNIQFAILERLRYPNVIREEIERLLENITVLAEAAALATSPALTDELVSHGELMSTLLFVEILRERDVQAQWFDVRKVMRTNDRFGRAEPDIAALAELAALQLLPRLNEGLVITQGFIGSENKGRTTTLGRGGSDYTAALLAEALHASRVDIWTDVPGIYTTDPRVVSAAKRIDEIAFAEAAEMATFGAKVLHPATLLPAVRSDIPVFVGSSKDPRAGGTLVCNKTENPPLFRALALRRNQTLLTLHSLNMLHSRGFLAEVFGILARHNISVDLITMSEVSVALILDTTGSTSTGDTLLTQSLLMELSALCRVEVEEGLALVALIGNDLSKACGVGKEVFGVLEPFNIRMICYGASSHNLCFLVPGEDAEQVVQKLHSNLFESEQ ID NO: 28 (Streptomyces lividans aspartokinase III polypeptide sequence, S-LysC) MGLVVQKYGGSSVADAEGIKRVAKRIVEAKKNGNQVVAVVSAMGDTTDELIDLAEQVSPIPAGRELDMLLTAGERISMALLAMAIKNLGHEAQSFTGSQAGVITDSVHNKARIIDVTPGRIRTSVDEGNVAIVAGFQGVSQDSKDITTLGRGGSDTTAVALAAALDADVCEIYTDVDGVFTADPRVVPKAKKIDWISFEDMLELAASGSKVLLHRCVEYARRYNIPIHVRSSFSGLQGTWVSSEPIKQGEKHVEQALISGVAHDTSEAKVTVVGVPDKPGEAAAIFRAIADAQVNIDMVVQNVSAASTGLTDISFTLPKSEGRKAIDALEKNRPGIGFDSLRYDDQIGKISLVGAGMKSNPGVTADFFTALSDAGVNIELISTSEIRISVVTRKDDVNEAVRAVHTAFGLDSD SDEAVVYGGTGRSEQ ID NO: 29 (Escherichia coli tetA polynucleotide sequence, nucleotides 1-558, tetA (nt 1-558)) ATGAAATCTAACAATGCGCTCATCGTCATCCTCGGCACCGTCACCCTGGATGCTGTAGGCATAGGCTTGGTTATGCCGGTACTGCCGGGCCTCTTGCGGGATATCGTCCATTCCGACAGCATCGCCAGTCACTATGGCGTGCTGCTAGCGCTATATGCGTTGATGCAATTTCTATGCGCACCCGTTCTCGGAGCACTGTCCGACCGCTTTGGCCGCCGCCCAGTCCTGCTCGCTTCGCTACTTGGAGCCACTATCGACTACGCGATCATGGCGACCACACCCGTCCTGTGGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATTAAGGGAGAGCGTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGCATGGCGGCCGACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCGGCATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCATTGGACCGCTGATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGASEQ ID NO: 30 (Escherichia coli TetA polypeptide sequence, amino acids 1-185, TetA (aa1-185)) MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHYGVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRIVAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPFLAAAVLNGLNLLLGCFLMQESHSEQ ID NO: 31 (Escherichia coli tetA polynucleotide sequence,nucleotides 1-291, tetA (nt 1-291))ATGAAATCTAACAATGCGCTCATCGTCATCCTCGGCACCGTCACCCTGGATGCTGTAGGCATAGGCTTGGTTATGCCGGTACTGCCGGGCCTCTTGCGGGATATCGTCCATTCCGACAGCATCGCCAGTCACTATGGCGTGCTGCTAGCGCTATATGCGTTGATGCAATTTCTATGCGCACCCGTTCTCGGAGCACTGTCCGACCGCTTTGGCCGCCGCCCAGTCCTGCTCGCTTCGCTACTTGGAGCCACTATCGACTACGCGATCATGGCGACCACACCCGTCCTGTAAGGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGCATGGCGGCCGACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCGGCATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCATTGGACCGCTGATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGASEQ ID NO: 32 (Escherichia coli TetA polypeptide sequence, aminoacids 1-96, TetA (aa1-96))MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHYGVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLSEQ ID NO: 33 (Escherichia coli CadA polypeptide sequence, aminoacids 1-565, CadA (aa1-565))MNVIAILNHMGVYFKEEPIRELHRALERLNFQIVYPNDRDDLLKLIENNA RLCGVIFDWDKYNLELCEEISKMNENLPLYAFANTYSTLDVSLNDLRLQI SFFEYALGAAEDIANKIKQTTDEYINTILPPLTKALFKYVREGKYTFCTP GH MGGTAFQKSPVGSLFYDFFGPNTMKSDISISVSELGSLLDHSGPHKEA EQYIARVFNADRSYMVTNGTSTANKIVGMYSAPAGSTILIDRNCHKSLTH LMMMSDVTPIYFRPTRNAYGILGGIPQSEFQHATIAKRVKETPNATWPVH AVITNSTYDGLLYNTDFIKKTLDVKSIHFDSAWVPYTNFSPIYEGKCGMS GGRVEGKVIYETQSTHKLLAAFSQASMIHVKGDVNEETFNEAYMMHTTTS PHYGIVASTETAAAMMKGNAGKRLINGSIERAIKFRKEIKRLRTESDGWF FDVWQPDHIDTTECWPLRSDSTWHGFKNIDNEHMYLDPIKVTLLTPGMEK DGTMSDFGIPASIVAKYLDEHGIVVEKTGPYNLLFLFSIGIDKTKALSLL  RALTDFKRAFDLNLR SEQ ID NO: 34 (ldc2-co1 C332G DNA sequence) ATGTACAAAGATCTGAAATTCCCTGTTCTGATTGTACACCGCGATATCAAGGCGGACACGGTAGCCGGCGAGCGTGTTCGCGGTATTGCCCACGAACTCGAACAAGACGGCTTTAGCATTCTCTCTACGGCGTCTTCTGCGGAAGGCCGCATTGTGGCTAGCACGCACCACGGTCTCGCCTGCATCCTCGTGGCAGCTGAGGGTGCGGGTGAGAATCAGCGTCTGCTCCAAGACGTGGTTGAGCTGATCCGTGTAGCTCGCGTCCGTGCGCCACAGCTCCCGATCTTCGCGCTGGGCGAACAGGTGACTATTGAAAACGCGCCTGCCGAATGTATGGCCGACCTGCACCAGCTCCGCGGCATTCTGTATCTCTTCGAGGATACTGTCCCGTTCCTGGCACGTCAGGTTGCACGCGCAGCGCGTAACTACCTCGCTGGCCTCCTCCCGCCATTCTTCCGTGCACTCGTGGAGCACACGGCCCAAAGCAATTACTCTTGGCACACCCCGGGTCACGGTGGTGGTGTCGCTTACCGTAAATCTCCGGTAGGTCAAGCTTTCCACCAGTTCTTTGGCGAGAATACCCTCCGCTCTGACCTGTCTGTTAGCGTTCCAGAGCTGGGCAGCCTGCTGGATCACACTGGCCCTCTCGCGGAAGCAGAGGATCGTGCCGCTCGCAATTTCGGTGCGGACCACACCTTCTTTGTCATCAATGGTACCTCTACTGCGAACAAAATCGTTTGGCACTCTATGGTTGGTCGCGAGGACCTGGTGCTGGTCGATCGTAACTGTCACAAATCTATTCTGCACTCCATTATCATGACGGGTGCTATCCCACTGTACCTGACTCCGGAACGCAACGAACTGGGTATTATCGGCCCTATTCCACTCTCCGAGTTTTCTAAACAATCTATCGCAGCAAAAATTGCCGCCTCCCCACTCGCGCGTGGTCGTGAACCGAAAGTTAAACTGGCTGTCGTTACCAACTCTACCTATGACGGTCTGTGTTACAACGCGGAACTGATCAAACAAACCCTCGGCGACTCTGTCGAGGTACTGCATTTCGACGAGGCTTGGTATGCTTATGCGGCGTTTCACGAGTTCTACGACGGCCGCTACGGTATGGGCACTTCTCGTTCCGAAGAGGGTCCGCTGGTCTTTGCTACCCATTCTACCCACAAGATGCTCGCGGCTTTTTCCCAAGCTAGCATGATTCACGTTCAGGATGGTGGTACGCGCAAGCTGGACGTCGCCCGCTTTAACGAAGCCTTTATGATGCACATCAGCACCTCTCCACAGTACGGCATCATTGCGTCTCTCGATGTCGCAAGCGCTATGATGGAAGGTCCTGCCGGTCGTAGCCTGATCCAAGAGACGTTCGATGAGGCGCTGTCCTTCCGTCGTGCTCTGGCGAATGTCCGTCAGAACCTGGACCGTAATGATTGGTGGTTCGGTGTCTGGCAACCGGAGCAGGTTGAGGGCACCGACCAGGTAGGTACTCACGACTGGGTTCTCGAGCCTAGCGCGGACTGGCATGGTTTTGGTGACATTGCGGAGGATTACGTTCTCCTCGATCCTATCAAAGTTACCCTGACCACCCCAGGTCTGAGCGCTGGCGGTAAACTCTCTGAACAAGGCATCCCGGCAGCTATCGTTAGCCGTTTCCTGTGGGAACGTGGTCTGGTGGTCGAGAAAACGGGTCTGTACTCTTTCCTGGTTCTGTTCTCCATGGGTATCACGAAAGGCAAATGGTCTACTCTGGTTACCGAGCTGCTCGAATTCAAACGCTGTTACGACGCGAATCTGCCACTCCTGGATGTGCTGCCTTCTGTAGCGCAGGCGGGTGGTAAACGCTATAACGGTGTAGGTCTGCGTGATCTGTCCGATGCCATGCACGCTTCTTATCGTGACAATGCCACGGCGAAGGCCATGAAGCGTATGTATACGGTGCTCCCGGAAGTAGCCATGCGCCCGTCCGAAGCTTATGATAAGCTCGTACGCGGTGAAGTCGAAGCTGTTCCTATTGCACGTCTCGAGGGTCGTATTGCGGCGGTTATGCTGGTTCCGTACCCGCCAGGTATCCCGCTCATTATGCCGGGTGAACGTTTTACTGAAGCTACCCGCTCCATTCTGGACTATCTGGAGTTTGCCCGTACCTTCGAGCGCGCGTTCCCGGGCTTTGACTCTGATGTTCACGGCCTCCAACATCAAGATGGCCCGTCTGGCCGTTGTTATACCGTTGAATGCATCAAGGAATAASEQ ID NO: 35 (ldc2-co1A785C DNA sequence)ATGTACAAAGATCTGAAATTCCCTGTTCTGATTGTACACCGCGATATCAAGGCGGACACGGTAGCCGGCGAGCGTGTTCGCGGTATTGCCCACGAACTCGAACAAGACGGCTTTAGCATTCTCTCTACGGCGTCTTCTGCGGAAGGCCGCATTGTGGCTAGCACGCACCACGGTCTCGCCTGCATCCTCGTGGCAGCTGAGGGTGCGGGTGAGAATCAGCGTCTGCTCCAAGACGTGGTTGAGCTGATCCGTGTAGCTCGCGTCCGTGCGCCACAGCTCCCGATCTTCGCGCTGGGCGAACAGGTGACTATTGAAAACGCGCCTGCCGAATCTATGGCCGACCTGCACCAGCTCCGCGGCATTCTGTATCTCTTCGAGGATACTGTCCCGTTCCTGGCACGTCAGGTTGCACGCGCAGCGCGTAACTACCTCGCTGGCCTCCTCCCGCCATTCTTCCGTGCACTCGTGGAGCACACGGCCCAAAGCAATTACTCTTGGCACACCCCGGGTCACGGTGGTGGTGTCGCTTACCGTAAATCTCCGGTAGGTCAAGCTTTCCACCAGTTCTTTGGCGAGAATACCCTCCGCTCTGACCTGTCTGTTAGCGTTCCAGAGCTGGGCAGCCTGCTGGATCACACTGGCCCTCTCGCGGAAGCAGAGGATCGTGCCGCTCGCAATTTCGGTGCGGACCACACCTTCTTTGTCATCAATGGTACCTCTACTGCGAACAAAATCGTTTGGCACTCTATGGTTGGTCGCGAGGACCTGGTGCTGGTCGATCGTACCTGTCACAAATCTATTCTGCACTCCATTATCATGACGGGTGCTATCCCACTGTACCTGACTCCGGAACGCAACGAACTGGGTATTATCGGCCCTATTCCACTCTCCGAGTTTTCTAAACAATCTATCGCAGCAAAAATTGCCGCCTCCCCACTCGCGCGTGGTCGTGAACCGAAAGTTAAACTGGCTGTCGTTACCAACTCTACCTATGACGGTCTGTGTTACAACGCGGAACTGATCAAACAAACCCTCGGCGACTCTGTCGAGGTACTGCATTTCGACGAGGCTTGGTATGCTTATGCGGCGTTTCACGAGTTCTACGACGGCCGCTACGGTATGGGCACTTCTCGTTCCGAAGAGGGTCCGCTGGTCTTTGCTACCCATTCTACCCACAAGATGCTCGCGGCTTTTTCCCAAGCTAGCATGATTCACGTTCAGGATGGTGGTACGCGCAAGCTGGACGTCGCCCGCTTTAACGAAGCCTTTATGATGCACATCAGCACCTCTCCACAGTACGGCATCATTGCGTCTCTCGATGTCGCAAGCGCTATGATGGAAGGTCCTGCCGGTCGTAGCCTGATCCAAGAGACGTTCGATGAGGCGCTGTCCTTCCGTCGTGCTCTGGCGAATGTCCGTCAGAACCTGGACCGTAATGATTGGTGGTTCGGTGTCTGGCAACCGGAGCAGGTTGAGGGCACCGACCAGGTAGGTACTCACGACTGGGTTCTCGAGCCTAGCGCGGACTGGCATGGTTTTGGTGACATTGCGGAGGATTACGTTCTCCTCGATCCTATCAAAGTTACCCTGACCACCCCAGGTCTGAGCGCTGGCGGTAAACTCTCTGAACAAGGCATCCCGGCAGCTATCGTTAGCCGTTTCCTGTGGGAACGTGGTCTGGTGGTCGAGAAAACGGGTCTGTACTCTTTCCTGGTTCTGTTCTCCATGGGTATCACGAAAGGCAAATGGTCTACTCTGGTTACCGAGCTGCTCGAATTCAAACGCTGTTACGACGCGAATCTGCCACTCCTGGATGTGCTGCCTTCTGTAGCGCAGGCGGGTGGTAAACGCTATAACGGTGTAGGTCTGCGTGATCTGTCCGATGCCATGCACGCTTCTTATCGTGACAATGCCACGGCGAAGGCCATGAAGCGTATGTATACGGTGCTCCCGGAAGTAGCCATGCGCCCGTCCGAAGCTTATGATAAGCTCGTACGCGGTGAAGTCGAAGCTGTTCCTATTGCACGTCTCGAGGGTCGTATTGCGGCGGTTATGCTGGTTCCGTACCCGCCAGGTATCCCGCTCATTATGCCGGGTGAACGTTTTACTGAAGCTACCCGCTCCATTCTGGACTATCTGGAGTTTGCCCGTACCTTCGAGCGCGCGTTCCCGGGCTTTGACTCTGATGTTCACGGCCTCCAACATCAAGATGGCCCGTCTGGCCGTTGTTATACCGTTGAATGCATCAAGGAATAASEQ ID NO: 36 (ldc2-co1 A795C DNA sequence)ATGTACAAAGATCTGAAATTCCCTGTTCTGATTGTACACCGCGATATCAAGGCGGACACGGTAGCCGGCGAGCGTGTTCGCGGTATTGCCCACGAACTCGAACAAGACGGCTTTAGCATTCTCTCTACGGCGTCTTCTGCGGAAGGCCGCATTGTGGCTAGCACGCACCACGGTCTCGCCTGCATCCTCGTGGCAGCTGAGGGTGCGGGTGAGAATCAGCGTCTGCTCCAAGACGTGGTTGAGCTGATCCGTGTAGCTCGCGTCCGTGCGCCACAGCTCCCGATCTTCGCGCTGGGCGAACAGGTGACTATTGAAAACGCGCCTGCCGAATCTATGGCCGACCTGCACCAGCTCCGCGGCATTCTGTATCTCTTCGAGGATACTGTCCCGTTCCTGGCACGTCAGGTTGCACGCGCAGCGCGTAACTACCTCGCTGGCCTCCTCCCGCCATTCTTCCGTGCACTCGTGGAGCACACGGCCCAAAGCAATTACTCTTGGCACACCCCGGGTCACGGTGGTGGTGTCGCTTACCGTAAATCTCCGGTAGGTCAAGCTTTCCACCAGTTCTTTGGCGAGAATACCCTCCGCTCTGACCTGTCTGTTAGCGTTCCAGAGCTGGGCAGCCTGCTGGATCACACTGGCCCTCTCGCGGAAGCAGAGGATCGTGCCGCTCGCAATTTCGGTGCGGACCACACCTTCTTTGTCATCAATGGTACCTCTACTGCGAACAAAATCGTTTGGCACTCTATGGTTGGTCGCGAGGACCTGGTGCTGGTCGATCGTAACTGTCACAACTCTATTCTGCACTCCATTATCATGACGGGTGCTATCCCACTGTACCTGACTCCGGAACGCAACGAACTGGGTATTATCGGCCCTATTCCACTCTCCGAGTTTTCTAAACAATCTATCGCAGCAAAAATTGCCGCCTCCCCACTCGCGCGTGGTCGTGAACCGAAAGTTAAACTGGCTGTCGTTACCAACTCTACCTATGACGGTCTGTGTTACAACGCGGAACTGATCAAACAAACCCTCGGCGACTCTGTCGAGGTACTGCATTTCGACGAGGCTTGGTATGCTTATGCGGCGTTTCACGAGTTCTACGACGGCCGCTACGGTATGGGCACTTCTCGTTCCGAAGAGGGTCCGCTGGTCTTTGCTACCCATTCTACCCACAAGATGCTCGCGGCTTTTTCCCAAGCTAGCATGATTCACGTTCAGGATGGTGGTACGCGCAAGCTGGACGTCGCCCGCTTTAACGAAGCCTTTATGATGCACATCAGCACCTCTCCACAGTACGGCATCATTGCGTCTCTCGATGTCGCAAGCGCTATGATGGAAGGTCCTGCCGGTCGTAGCCTGATCCAAGAGACGTTCGATGAGGCGCTGTCCTTCCGTCGTGCTCTGGCGAATGTCCGTCAGAACCTGGACCGTAATGATTGGTGGTTCGGTGTCTGGCAACCGGAGCAGGTTGAGGGCACCGACCAGGTAGGTACTCACGACTGGGTTCTCGAGCCTAGCGCGGACTGGCATGGTTTTGGTGACATTGCGGAGGATTACGTTCTCCTCGATCCTATCAAAGTTACCCTGACCACCCCAGGTCTGAGCGCTGGCGGTAAACTCTCTGAACAAGGCATCCCGGCAGCTATCGTTAGCCGTTTCCTGTGGGAACGTGGTCTGGTGGTCGAGAAAACGGGTCTGTACTCTTTCCTGGTTCTGTTCTCCATGGGTATCACGAAAGGCAAATGGTCTACTCTGGTTACCGAGCTGCTCGAATTCAAACGCTGTTACGACGCGAATCTGCCACTCCTGGATGTGCTGCCTTCTGTAGCGCAGGCGGGTGGTAAACGCTATAACGGTGTAGGTCTGCGTGATCTGTCCGATGCCATGCACGCTTCTTATCGTGACAATGCCACGGCGAAGGCCATGAAGCGTATGTATACGGTGCTCCCGGAAGTAGCCATGCGCCCGTCCGAAGCTTATGATAAGCTCGTACGCGGTGAAGTCGAAGCTGTTCCTATTGCACGTCTCGAGGGTCGTATTGCGGCGGTTATGCTGGTTCCGTACCCGCCAGGTATCCCGCTCATTATGCCGGGTGAACGTTTTACTGAAGCTACCCGCTCCATTCTGGACTATCTGGAGTTTGCCCGTACCTTCGAGCGCGCGTTCCCGGGCTTTGACTCTGATGTTCACGGCCTCCAACATCAAGATGGCCCGTCTGGCCGTTGTTATACCGTTGAATGCATCAAGGAATAASEQ ID NO: 37 (ldc2-co1 C332G/A785C DNA sequence)ATGTACAAAGATCTGAAATTCCCTGTTCTGATTGTACACCGCGATATCAAGGCGGACACGGTAGCCGGCGAGCGTGTTCGCGGTATTGCCCACGAACTCGAACAAGACGGCTTTAGCATTCTCTCTACGGCGTCTTCTGCGGAAGGCCGCATTGTGGCTAGCACGCACCACGGTCTCGCCTGCATCCTCGTGGCAGCTGAGGGTGCGGGTGAGAATCAGCGTCTGCTCCAAGACGTGGTTGAGCTGATCCGTGTAGCTCGCGTCCGTGCGCCACAGCTCCCGATCTTCGCGCTGGGCGAACAGGTGACTATTGAAAACGCGCCTGCCGAATGTATGGCCGACCTGCACCAGCTCCGCGGCATTCTGTATCTCTTCGAGGATACTGTCCCGTTCCTGGCACGTCAGGTTGCACGCGCAGCGCGTAACTACCTCGCTGGCCTCCTCCCGCCATTCTTCCGTGCACTCGTGGAGCACACGGCCCAAAGCAATTACTCTTGGCACACCCCGGGTCACGGTGGTGGTGTCGCTTACCGTAAATCTCCGGTAGGTCAAGCTTTCCACCAGTTCTTTGGCGAGAATACCCTCCGCTCTGACCTGTCTGTTAGCGTTCCAGAGCTGGGCAGCCTGCTGGATCACACTGGCCCTCTCGCGGAAGCAGAGGATCGTGCCGCTCGCAATTTCGGTGCGGACCACACCTTCTTTGTCATCAATGGTACCTCTACTGCGAACAAAATCGTTTGGCACTCTATGGTTGGTCGCGAGGACCTGGTGCTGGTCGATCGTACCTGTCACAAATCTATTCTGCACTCCATTATCATGACGGGTGCTATCCCACTGTACCTGACTCCGGAACGCAACGAACTGGGTATTATCGGCCCTATTCCACTCTCCGAGTTTTCTAAACAATCTATCGCAGCAAAAATTGCCGCCTCCCCACTCGCGCGTGGTCGTGAACCGAAAGTTAAACTGGCTGTCGTTACCAACTCTACCTATGACGGTCTGTGTTACAACGCGGAACTGATCAAACAAACCCTCGGCGACTCTGTCGAGGTACTGCATTTCGACGAGGCTTGGTATGCTTATGCGGCGTTTCACGAGTTCTACGACGGCCGCTACGGTATGGGCACTTCTCGTTCCGAAGAGGGTCCGCTGGTCTTTGCTACCCATTCTACCCACAAGATGCTCGCGGCTTTTTCCCAAGCTAGCATGATTCACGTTCAGGATGGTGGTACGCGCAAGCTGGACGTCGCCCGCTTTAACGAAGCCTTTATGATGCACATCAGCACCTCTCCACAGTACGGCATCATTGCGTCTCTCGATGTCGCAAGCGCTATGATGGAAGGTCCTGCCGGTCGTAGCCTGATCCAAGAGACGTTCGATGAGGCGCTGTCCTTCCGTCGTGCTCTGGCGAATGTCCGTCAGAACCTGGACCGTAATGATTGGTGGTTCGGTGTCTGGCAACCGGAGCAGGTTGAGGGCACCGACCAGGTAGGTACTCACGACTGGGTTCTCGAGCCTAGCGCGGACTGGCATGGTTTTGGTGACATTGCGGAGGATTACGTTCTCCTCGATCCTATCAAAGTTACCCTGACCACCCCAGGTCTGAGCGCTGGCGGTAAACTCTCTGAACAAGGCATCCCGGCAGCTATCGTTAGCCGTTTCCTGTGGGAACGTGGTCTGGTGGTCGAGAAAACGGGTCTGTACTCTTTCCTGGTTCTGTTCTCCATGGGTATCACGAAAGGCAAATGGTCTACTCTGGTTACCGAGCTGCTCGAATTCAAACGCTGTTACGACGCGAATCTGCCACTCCTGGATGTGCTGCCTTCTGTAGCGCAGGCGGGTGGTAAACGCTATAACGGTGTAGGTCTGCGTGATCTGTCCGATGCCATGCACGCTTCTTATCGTGACAATGCCACGGCGAAGGCCATGAAGCGTATGTATACGGTGCTCCCGGAAGTAGCCATGCGCCCGTCCGAAGCTTATGATAAGCTCGTACGCGGTGAAGTCGAAGCTGTTCCTATTGCACGTCTCGAGGGTCGTATTGCGGCGGTTATGCTGGTTCCGTACCCGCCAGGTATCCCGCTCATTATGCCGGGTGAACGTTTTACTGAAGCTACCCGCTCCATTCTGGACTATCTGGAGTTTGCCCGTACCTTCGAGCGCGCGTTCCCGGGCTTTGACTCTGATGTTCACGGCCTCCAACATCAAGATGGCCCGTCTGGCCGTTGTTATACCGTTGAATGCATCAAGGAATAASEQ ID NO: 38 (ldc2-co1 C332G/A795C DNA sequence)ATGTACAAAGATCTGAAATTCCCTGTTCTGATTGTACACCGCGATATCAAGGCGGACACGGTAGCCGGCGAGCGTGTTCGCGGTATTGCCCACGAACTCGAACAAGACGGCTTTAGCATTCTCTCTACGGCGTCTTCTGCGGAAGGCCGCATTGTGGCTAGCACGCACCACGGTCTCGCCTGCATCCTCGTGGCAGCTGAGGGTGCGGGTGAGAATCAGCGTCTGCTCCAAGACGTGGTTGAGCTGATCCGTGTAGCTCGCGTCCGTGCGCCACAGCTCCCGATCTTCGCGCTGGGCGAACAGGTGACTATTGAAAACGCGCCTGCCGAATGTATGGCCGACCTGCACCAGCTCCGCGGCATTCTGTATCTCTTCGAGGATACTGTCCCGTTCCTGGCACGTCAGGTTGCACGCGCAGCGCGTAACTACCTCGCTGGCCTCCTCCCGCCATTCTTCCGTGCACTCGTGGAGCACACGGCCCAAAGCAATTACTCTTGGCACACCCCGGGTCACGGTGGTGGTGTCGCTTACCGTAAATCTCCGGTAGGTCAAGCTTTCCACCAGTTCTTTGGCGAGAATACCCTCCGCTCTGACCTGTCTGTTAGCGTTCCAGAGCTGGGCAGCCTGCTGGATCACACTGGCCCTCTCGCGGAAGCAGAGGATCGTGCCGCTCGCAATTTCGGTGCGGACCACACCTTCTTTGTCATCAATGGTACCTCTACTGCGAACAAAATCGTTTGGCACTCTATGGTTGGTCGCGAGGACCTGGTGCTGGTCGATCGTAACTGTCACAACTCTATTCTGCACTCCATTATCATGACGGGTGCTATCCCACTGTACCTGACTCCGGAACGCAACGAACTGGGTATTATCGGCCCTATTCCACTCTCCGAGTTTTCTAAACAATCTATCGCAGCAAAAATTGCCGCCTCCCCACTCGCGCGTGGTCGTGAACCGAAAGTTAAACTGGCTGTCGTTACCAACTCTACCTATGACGGTCTGTGTTACAACGCGGAACTGATCAAACAAACCCTCGGCGACTCTGTCGAGGTACTGCATTTCGACGAGGCTTGGTATGCTTATGCGGCGTTTCACGAGTTCTACGACGGCCGCTACGGTATGGGCACTTCTCGTTCCGAAGAGGGTCCGCTGGTCTTTGCTACCCATTCTACCCACAAGATGCTCGCGGCTTTTTCCCAAGCTAGCATGATTCACGTTCAGGATGGTGGTACGCGCAAGCTGGACGTCGCCCGCTTTAACGAAGCCTTTATGATGCACATCAGCACCTCTCCACAGTACGGCATCATTGCGTCTCTCGATGTCGCAAGCGCTATGATGGAAGGTCCTGCCGGTCGTAGCCTGATCCAAGAGACGTTCGATGAGGCGCTGTCCTTCCGTCGTGCTCTGGCGAATGTCCGTCAGAACCTGGACCGTAATGATTGGTGGTTCGGTGTCTGGCAACCGGAGCAGGTTGAGGGCACCGACCAGGTAGGTACTCACGACTGGGTTCTCGAGCCTAGCGCGGACTGGCATGGTTTTGGTGACATTGCGGAGGATTACGTTCTCCTCGATCCTATCAAAGTTACCCTGACCACCCCAGGTCTGAGCGCTGGCGGTAAACTCTCTGAACAAGGCATCCCGGCAGCTATCGTTAGCCGTTTCCTGTGGGAACGTGGTCTGGTGGTCGAGAAAACGGGTCTGTACTCTTTCCTGGTTCTGTTCTCCATGGGTATCACGAAAGGCAAATGGTCTACTCTGGTTACCGAGCTGCTCGAATTCAAACGCTGTTACGACGCGAATCTGCCACTCCTGGATGTGCTGCCTTCTGTAGCGCAGGCGGGTGGTAAACGCTATAACGGTGTAGGTCTGCGTGATCTGTCCGATGCCATGCACGCTTCTTATCGTGACAATGCCACGGCGAAGGCCATGAAGCGTATGTATACGGTGCTCCCGGAAGTAGCCATGCGCCCGTCCGAAGCTTATGATAAGCTCGTACGCGGTGAAGTCGAAGCTGTTCCTATTGCACGTCTCGAGGGTCGTATTGCGGCGGTTATGCTGGTTCCGTACCCGCCAGGTATCCCGCTCATTATGCCGGGTGAACGTTTTACTGAAGCTACCCGCTCCATTCTGGACTATCTGGAGTTTGCCCGTACCTTCGAGCGCGCGTTCCCGGGCTTTGACTCTGATGTTCACGGCCTCCAACATCAAGATGGCCCGTCTGGCCGTTGTTATACCGTTGAATGCATCAAGGAATAASEQ ID NO: 39 (ldc2-co1 A785C/A795C DNA sequence)ATGTACAAAGATCTGAAATTCCCTGTTCTGATTGTACACCGCGATATCAAGGCGGACACGGTAGCCGGCGAGCGTGTTCGCGGTATTGCCCACGAACTCGAACAAGACGGCTTTAGCATTCTCTCTACGGCGTCTTCTGCGGAAGGCCGCATTGTGGCTAGCACGCACCACGGTCTCGCCTGCATCCTCGTGGCAGCTGAGGGTGCGGGTGAGAATCAGCGTCTGCTCCAAGACGTGGTTGAGCTGATCCGTGTAGCTCGCGTCCGTGCGCCACAGCTCCCGATCTTCGCGCTGGGCGAACAGGTGACTATTGAAAACGCGCCTGCCGAATCTATGGCCGACCTGCACCAGCTCCGCGGCATTCTGTATCTCTTCGAGGATACTGTCCCGTTCCTGGCACGTCAGGTTGCACGCGCAGCGCGTAACTACCTCGCTGGCCTCCTCCCGCCATTCTTCCGTGCACTCGTGGAGCACACGGCCCAAAGCAATTACTCTTGGCACACCCCGGGTCACGGTGGTGGTGTCGCTTACCGTAAATCTCCGGTAGGTCAAGCTTTCCACCAGTTCTTTGGCGAGAATACCCTCCGCTCTGACCTGTCTGTTAGCGTTCCAGAGCTGGGCAGCCTGCTGGATCACACTGGCCCTCTCGCGGAAGCAGAGGATCGTGCCGCTCGCAATTTCGGTGCGGACCACACCTTCTTTGTCATCAATGGTACCTCTACTGCGAACAAAATCGTTTGGCACTCTATGGTTGGTCGCGAGGACCTGGTGCTGGTCGATCGTACCTGTCACAACTCTATTCTGCACTCCATTATCATGACGGGTGCTATCCCACTGTACCTGACTCCGGAACGCAACGAACTGGGTATTATCGGCCCTATTCCACTCTCCGAGTTTTCTAAACAATCTATCGCAGCAAAAATTGCCGCCTCCCCACTCGCGCGTGGTCGTGAACCGAAAGTTAAACTGGCTGTCGTTACCAACTCTACCTATGACGGTCTGTGTTACAACGCGGAACTGATCAAACAAACCCTCGGCGACTCTGTCGAGGTACTGCATTTCGACGAGGCTTGGTATGCTTATGCGGCGTTTCACGAGTTCTACGACGGCCGCTACGGTATGGGCACTTCTCGTTCCGAAGAGGGTCCGCTGGTCTTTGCTACCCATTCTACCCACAAGATGCTCGCGGCTTTTTCCCAAGCTAGCATGATTCACGTTCAGGATGGTGGTACGCGCAAGCTGGACGTCGCCCGCTTTAACGAAGCCTTTATGATGCACATCAGCACCTCTCCACAGTACGGCATCATTGCGTCTCTCGATGTCGCAAGCGCTATGATGGAAGGTCCTGCCGGTCGTAGCCTGATCCAAGAGACGTTCGATGAGGCGCTGTCCTTCCGTCGTGCTCTGGCGAATGTCCGTCAGAACCTGGACCGTAATGATTGGTGGTTCGGTGTCTGGCAACCGGAGCAGGTTGAGGGCACCGACCAGGTAGGTACTCACGACTGGGTTCTCGAGCCTAGCGCGGACTGGCATGGTTTTGGTGACATTGCGGAGGATTACGTTCTCCTCGATCCTATCAAAGTTACCCTGACCACCCCAGGTCTGAGCGCTGGCGGTAAACTCTCTGAACAAGGCATCCCGGCAGCTATCGTTAGCCGTTTCCTGTGGGAACGTGGTCTGGTGGTCGAGAAAACGGGTCTGTACTCTTTCCTGGTTCTGTTCTCCATGGGTATCACGAAAGGCAAATGGTCTACTCTGGTTACCGAGCTGCTCGAATTCAAACGCTGTTACGACGCGAATCTGCCACTCCTGGATGTGCTGCCTTCTGTAGCGCAGGCGGGTGGTAAACGCTATAACGGTGTAGGTCTGCGTGATCTGTCCGATGCCATGCACGCTTCTTATCGTGACAATGCCACGGCGAAGGCCATGAAGCGTATGTATACGGTGCTCCCGGAAGTAGCCATGCGCCCGTCCGAAGCTTATGATAAGCTCGTACGCGGTGAAGTCGAAGCTGTTCCTATTGCACGTCTCGAGGGTCGTATTGCGGCGGTTATGCTGGTTCCGTACCCGCCAGGTATCCCGCTCATTATGCCGGGTGAACGTTTTACTGAAGCTACCCGCTCCATTCTGGACTATCTGGAGTTTGCCCGTACCTTCGAGCGCGCGTTCCCGGGCTTTGACTCTGATGTTCACGGCCTCCAACATCAAGATGGCCCGTCTGGCCGTTGTTATACCGTTGAATGCATCAAGGAATAASEQ ID NO: 40 (ldc2-co1 C332G/A785C/A795C DNA sequence) ATGTACAAAGATCTGAAATTCCCTGTTCTGATTGTACACCGCGATATCAAGGCGGACACGGTAGCCGGCGAGCGTGTTCGCGGTATTGCCCACGAACTCGAACAAGACGGCTTTAGCATTCTCTCTACGGCGTCTTCTGCGGAAGGCCGCATTGTGGCTAGCACGCACCACGGTCTCGCCTGCATCCTCGTGGCAGCTGAGGGTGCGGGTGAGAATCAGCGTCTGCTCCAAGACGTGGTTGAGCTGATCCGTGTAGCTCGCGTCCGTGCGCCACAGCTCCCGATCTTCGCGCTGGGCGAACAGGTGACTATTGAAAACGCGCCTGCCGAATGTATGGCCGACCTGCACCAGCTCCGCGGCATTCTGTATCTCTTCGAGGATACTGTCCCGTTCCTGGCACGTCAGGTTGCACGCGCAGCGCGTAACTACCTCGCTGGCCTCCTCCCGCCATTCTTCCGTGCACTCGTGGAGCACACGGCCCAAAGCAATTACTCTTGGCACACCCCGGGTCACGGTGGTGGTGTCGCTTACCGTAAATCTCCGGTAGGTCAAGCTTTCCACCAGTTCTTTGGCGAGAATACCCTCCGCTCTGACCTGTCTGTTAGCGTTCCAGAGCTGGGCAGCCTGCTGGATCACACTGGCCCTCTCGCGGAAGCAGAGGATCGTGCCGCTCGCAATTTCGGTGCGGACCACACCTTCTTTGTCATCAATGGTACCTCTACTGCGAACAAAATCGTTTGGCACTCTATGGTTGGTCGCGAGGACCTGGTGCTGGTCGATCGTACCTGTCACAACTCTATTCTGCACTCCATTATCATGACGGGTGCTATCCCACTGTACCTGACTCCGGAACGCAACGAACTGGGTATTATCGGCCCTATTCCACTCTCCGAGTTTTCTAAACAATCTATCGCAGCAAAAATTGCCGCCTCCCCACTCGCGCGTGGTCGTGAACCGAAAGTTAAACTGGCTGTCGTTACCAACTCTACCTATGACGGTCTGTGTTACAACGCGGAACTGATCAAACAAACCCTCGGCGACTCTGTCGAGGTACTGCATTTCGACGAGGCTTGGTATGCTTATGCGGCGTTTCACGAGTTCTACGACGGCCGCTACGGTATGGGCACTTCTCGTTCCGAAGAGGGTCCGCTGGTCTTTGCTACCCATTCTACCCACAAGATGCTCGCGGCTTTTTCCCAAGCTAGCATGATTCACGTTCAGGATGGTGGTACGCGCAAGCTGGACGTCGCCCGCTTTAACGAAGCCTTTATGATGCACATCAGCACCTCTCCACAGTACGGCATCATTGCGTCTCTCGATGTCGCAAGCGCTATGATGGAAGGTCCTGCCGGTCGTAGCCTGATCCAAGAGACGTTCGATGAGGCGCTGTCCTTCCGTCGTGCTCTGGCGAATGTCCGTCAGAACCTGGACCGTAATGATTGGTGGTTCGGTGTCTGGCAACCGGAGCAGGTTGAGGGCACCGACCAGGTAGGTACTCACGACTGGGTTCTCGAGCCTAGCGCGGACTGGCATGGTTTTGGTGACATTGCGGAGGATTACGTTCTCCTCGATCCTATCAAAGTTACCCTGACCACCCCAGGTCTGAGCGCTGGCGGTAAACTCTCTGAACAAGGCATCCCGGCAGCTATCGTTAGCCGTTTCCTGTGGGAACGTGGTCTGGTGGTCGAGAAAACGGGTCTGTACTCTTTCCTGGTTCTGTTCTCCATGGGTATCACGAAAGGCAAATGGTCTACTCTGGTTACCGAGCTGCTCGAATTCAAACGCTGTTACGACGCGAATCTGCCACTCCTGGATGTGCTGCCTTCTGTAGCGCAGGCGGGTGGTAAACGCTATAACGGTGTAGGTCTGCGTGATCTGTCCGATGCCATGCACGCTTCTTATCGTGACAATGCCACGGCGAAGGCCATGAAGCGTATGTATACGGTGCTCCCGGAAGTAGCCATGCGCCCGTCCGAAGCTTATGATAAGCTCGTACGCGGTGAAGTCGAAGCTGTTCCTATTGCACGTCTCGAGGGTCGTATTGCGGCGGTTATGCTGGTTCCGTACCCGCCAGGTATCCCGCTCATTATGCCGGGTGAACGTTTTACTGAAGCTACCCGCTCCATTCTGGACTATCTGGAGTTTGCCCGTACCTTCGAGCGCGCGTTCCCGGGCTTTGACTCTGATGTTCACGGCCTCCAACATCAAGATGGCCCGTCTGGCCGTTGTTATACCGTTGAATGCATCAAGGAATAASEQ ID NO: 41 (cadA DNA sequence) ATGAACGTTATTGCAATATTGAATCACATGGGGGTTTATTTTAAAGAAGAACCCATCCGTGAACTTCATCGCGCGCTTGAACGTCTGAACTTCCAGATTGTTTACCCGAACGACCGTGACGACTTATTAAAACTGATCGAAAACAATGCGCGTCTGTGCGGCGTTATTTTTGACTGGGATAAATATAATCTCGAGCTGTGCGAAGAAATTAGCAAAATGAACGAGAACCTGCCGTTGTACGCGTTCGCTAATACGTATTCCACTCTCGATGTAAGCCTGAATGACCTGCGTTTACAGATTAGCTTCTTTGAATATGCGCTGGGTGCTGCTGAAGATATTGCTAATAAGATCAAGCAGACCACTGACGAATATATCAACACTATTCTGCCTCCGCTGACTAAAGCACTGTTTAAATATGTTCGTGAAGGTAAATATACTTTCTGTACTCCTGGTCACATGGGCGGTACTGCATTCCAGAAAAGCCCGGTAGGTAGCCTGTTCTATGATTTCTTTGGTCCGAATACCATGAAATCTGATATTTCCATTTCAGTATCTGAACTGGGTTCTCTGCTGGATCACAGTGGTCCACACAAAGAAGCAGAACAGTATATCGCTCGCGTCTTTAACGCAGACCGCAGCTACATGGTGACCAACGGTACTTCCACTGCGAACAAAATTGTTGGTATGTACTCTGCTCCAGCAGGCAGCACCATTCTGATTGACCGTAACTGCCACAAATCGCTGACCCACCTGATGATGATGAGCGATGTTACGCCAATCTATTTCCGCCCGACCCGTAACGCTTACGGTATTCTTGGTGGTATCCCACAGAGTGAATTCCAGCACGCTACCATTGCTAAGCGCGTGAAAGAAACACCAAACGCAACCTGGCCGGTACATGCTGTAATTACCAACTCTACCTATGATGGTCTGCTGTACAACACCGACTTCATCAAGAAAACACTGGATGTGAAATCCATCCACTTTGACTCCGCGTGGGTGCCTTACACCAACTTCTCACCGATTTACGAAGGTAAATGCGGTATGAGCGGTGGCCGTGTAGAAGGGAAAGTGATTTACGAAACCCAGTCCACTCACAAACTGCTGGCGGCGTTCTCTCAGGCTTCCATGATCCACGTTAAAGGTGACGTAAACGAAGAAACCTTTAACGAAGCCTACATGATGCACACCACCACTTCTCCGCACTACGGTATCGTGGCGTCCACTGAAACCGCTGCGGCGATGATGAAAGGCAATGCAGGTAAGCGTCTGATCAACGGTTCTATTGAACGTGCGATCAAATTCCGTAAAGAGATCAAACGTCTGAGAACGGAATCTGATGGCTGGTTCTTTGATGTATGGCAGCCGGATCATATCGATACGACTGAATGCTGGCCGCTGCGTTCTGACAGCACCTGGCACGGCTTCAAAAACATCGATAACGAGCACATGTATCTTGACCCGATCAAAGTCACCCTGCTGACTCCGGGGATGGAAAAAGACGGCACCATGAGCGACTTTGGTATTCCGGCCAGCATCGTGGCGAAATACCTCGACGAACATGGCATCGTTGTTGAGAAAACCGGTCCGTATAACCTGCTGTTCCTGTTCAGCATCGGTATCGATAAGACCAAAGCACTGAGCCTGCTGCGTGCTCTGACTGACTTTAAACGTGCGTTCGACCTGAACCTGCGTGTGAAAAACATGCTGCCGTCTCTGTATCGTGAAGATCCTGAATTCTATGAAAACATGCGTATTCAGGAACTGGCTCAGAATATCCACAAACTGATTGTTCACCACAATCTGCCGGATCTGATGTATCGCGCATTTGAAGTGCTGCCGACGATGGTAATGACTCCGTATGCTGCATTCCAGAAAGAGCTGCACGGTATGACCGAAGAAGTTTACCTCGACGAAATGGTAGGTCGTATTAACGCCAATATGATCCTTCCGTACCCGCCGGGAGTTCCTCTGGTAATGCCGGGTGAAATGATCACCGAAGAAAGCCGTCCGGTTCTGGAGTTCCTGCAGATGCTGTGTGAAATCGGCGCTCACTATCCGGGCTTTGAAACCGATATTCACGGTGCATACCGTCAGGCTGATGGCCGCTATACCGTTAAGGTATTGAAAGAAGAAAGCAAAAAATAA SEQ ID NO: 42 (ldcC DNA sequence) ATGAACATCATTGCCATTATGGGACCGCATGGCGTCTTTTATAAAGATGAGCCCATCAAAGAACTGGAGTCGGCGCTGGTGGCGCAAGGCTTTCAGATTATCTGGCCACAAAACAGCGTTGATTTGCTGAAATTTATCGAGCATAACCCTCGAATTTGCGGCGTGATTTTTGACTGGGATGAGTACAGTCTCGATTTATGTAGCGATATCAATCAGCTTAATGAATATCTCCCGCTTTATGCCTTCATCAACACCCACTCGACGATGGATGTCAGCGTGCAGGATATGCGGATGGCGCTCTGGTTTTTTGAATATGCGCTGGGGCAGGCGGAAGATATCGCCATTCGTATGCGTCAGTACACCGACGAATATCTTGATAACATTACACCGCCGTTCACGAAAGCCTTGTTTACCTACGTCAAAGAGCGGAAGTACACCTTTTGTACGCCGGGGCATATGGGCGGCACCGCATATCAAAAAAGCCCGGTTGGCTGTCTGTTTTATGATTTTTTCGGCGGGAATACTCTTAAGGCTGATGTCTCTATTTCGGTCACCGAGCTTGGTTCGTTGCTCGACCACACCGGGCCACACCTGGAAGCGGAAGAGTACATCGCGCGGACTTTTGGCGCGGAACAGAGTTATATCGTTACCAACGGAACATCGACGTCGAACAAAATTGTGGGTATGTACGCCGCGCCATCCGGCAGTACGCTGTTGATCGACCGCAATTGTCATAAATCGCTGGCGCATCTGTTGATGATGAACGATGTAGTGCCAGTCTGGCTGAAACCGACGCGTAATGCGTTGGGGATTCTTGGTGGGATCCCGCGCCGTGAATTTACTCGCGACAGCATCGAAGAGAAAGTCGCTGCTACCACGCAAGCACAATGGCCGGTTCATGCGGTGATCACCAACTCCACCTATGATGGCTTGCTCTACAACACCGACTGGATCAAACAGACGCTGGATGTCCCGTCGATTCACTTCGATTCTGCCTGGGTGCCGTACACCCATTTTCATCCGATCTACCAGGGTAAAAGTGGTATGAGCGGCGAGCGTGTTGCGGGAAAAGTGATCTTCGAAACGCAATCGACCCACAAAATGCTGGCGGCGTTATCGCAGGCTTCGCTGATCCACATTAAAGGCGAGTATGACGAAGAGGCCTTTAACGAAGCCTTTATGATGCATACCACCACCTCGCCCAGTTATCCCATTGTTGCTTCGGTTGAGACGGCGGCGGCGATGCTGCGTGGTAATCCGGGCAAACGGCTGATTAACCGTTCAGTAGAACGAGCTCTGCATTTTCGCAAAGAGGTCCAGCGGCTGCGGGAAGAGTCTGACGGTTGGTTTTTCGATATCTGGCAACCGCCGCAGGTGGATGAAGCCGAATGCTGGCCCGTTGCGCCTGGCGAACAGTGGCACGGCTTTAACGATGCGGATGCCGATCATATGTTTCTCGATCCGGTTAAAGTCACTATTTTGACACCGGGGATGGACGAGCAGGGCAATATGAGCGAGGAGGGGATCCCGGCGGCGCTGGTAGCAAAATTCCTCGACGAACGTGGGATCGTAGTAGAGAAAACCGGCCCTTATAACCTGCTGTTTCTCTTTAGTATTGGCATCGATAAAACCAAAGCAATGGGATTATTGCGTGGGTTGACGGAATTCAAACGCTCTTACGATCTCAACCTGCGGATCAAAAATATGCTACCCGATCTCTATGCAGAAGATCCCGATTTCTACCGCAATATGCGTATTCAGGATCTGGCACAAGGGATCCATAAGCTGATTCGTAAACACGATCTTCCCGGTTTGATGTTGCGGGCATTCGATACTTTGCCGGAGATGATCATGACGCCACATCAGGCATGGCAACGACAAATTAAAGGCGAAGTAGAAACCATTGCGCTGGAACAACTGGTCGGTAGAGTATCGGCAAATATGATCCTGCCTTATCCACCGGGCGTACCGCTGTTGATGCCTGGAGAAATGCTGACCAAAGAGAGCCGCACAGTACTCGATTTTCTACTGATGCTTTGTTCCGTCGGGCAACATTACCCCGGTTTTGAAACGGATATTCACGGCGCGAAACAGGACGAAGACGGCGTTTACCGCGTACGAGTCCTAAAAATGGCGGGATAA

REFERENCES

The references, patents and published patent applications listed below,and all references cited in the specification above are herebyincorporated by reference in their entirety, as if fully set forthherein.

-   1. Wertz et al. “Chimeric nature of two plasmids of Hafnia alvei    encoding the bacteriocins alveicins A and B.” Journal of    Bacteriology, (2004) 186: 1598-1605).

1-30. (canceled)
 31. A product, which is one of the following productsI) through V): I) a first polypeptide comprising a mutant of the aminoacid sequence SEQ ID NO: 2 (TetA) and fragments thereof; II) anon-naturally occurring first polynucleotide encoding the firstpolypeptide of the product I); III) a first expression plasmid vectorcomprising: one or more first polynucleotides encoding a firstpolypeptide comprising a tetracycline efflux pump polypeptide, afragment thereof, or a mutant thereof, and a backbone plasmid capable ofautonomous replication in a host cell, wherein the first expressionplasmid vector is used for production of lysine or a lysine-derivedproduct; IV) a transformant comprising one or more the first expressionplasmid vectors of the product III) in a host cell; V) a mutant hostcell comprising one or more first polynucleotides integrated into achromosome of a host cell, wherein the first polynucleotide encodes afirst polypeptide comprising a tetracycline efflux pump polypeptide, afragment thereof or a mutant thereof.
 32. A product of claim 31, whichis I) the first polypeptide, wherein the mutant of TetA is selected fromthe group consisting of amino acid sequences of SEQ ID NO: 30 (TetA(aa1-185)) and SEQ ID NO: 32 (TetA (aa1-96)).
 33. A product of claim 31,which is II) the non-naturally occurring first polynucleotide, whereinthe first polynucleotide sequence is selected from the group consistingof SEQ ID NO: 29 (tetA (nt 1-558)) and SEQ ID NO: 31 (tetA (nt 1-291)).34. A product of claim 31, which is II) the non-naturally occurringpolynucleotide, wherein the first polynucleotide sequence has been codonoptimized for optimal polypeptide expression in E. coli.
 35. A productof claim 31, which is III) the first expression plasmid vector, furthercomprising: one or more second polynucleotides independently selectedfrom the group consisting of: a third polynucleotide encoding a thirdpolypeptide comprising a lysine decarboxylase polypeptide, a fragmentthereof or a mutant thereof, and a fourth polynucleotide encoding afourth polypeptide comprising a lysine biosynthesis polypeptide, afragment thereof or a mutant thereof.
 36. A product of claim 31, whichis IV) the transformant, which further comprises one or more secondexpression plasmid vectors comprising one or more fifth polynucleotidesindependently selected from the group consisting of a firstpolynucleotide encoding a first polypeptide comprising a tetracyclineefflux pump polypeptide, a fragment thereof or a mutant thereof, a thirdpolynucleotide encoding a third polypeptide comprising a lysinedecarboxylase polypeptide, a fragment thereof or a mutant thereof, and afourth polynucleotide encoding a fourth polypeptide comprising a lysinebiosynthesis polypeptide, a fragment thereof or a mutant thereof; and abackbone plasmid capable of autonomous replication in a host cell,wherein the one or more second expression plasmid vectors are used forproduction of lysine or a lysine-derived product.
 37. A product of claim31, which is IV) the transformant, wherein the backbone plasmid is an E.coli expression plasmid vector.
 38. A product of claim 31, which is III)the first expression plasmid vector, wherein the backbone plasmid is anE. coli expression plasmid vector.
 39. A product of claim 37, whereinthe backbone plasmid is selected from the group consisting of pUC18,pUC19, pBR322, pACYC, pET, pSC101 and any derived plasmids thereof. 40.A product of claim 38, wherein the backbone plasmid is selected from thegroup consisting of pUC18, pUC19, pBR322, pACYC, pET, pSC101 and anyderived plasmids thereof.
 41. A product of claim 31, which is IV) thetransformant, wherein the expression plasmid vector further comprisesone or more sixth polynucleotides encoding a sixth polypeptidecomprising an antibiotic resistance protein, a fragment thereof, ormutant thereof.
 42. A product of claim 31, which is III) the firstexpression plasmid vector, wherein the expression plasmid vector furthercomprises one or more sixth polynucleotides encoding a sixth polypeptidecomprising an antibiotic resistance protein, a fragment thereof, ormutant thereof.
 43. A product of claim 41, wherein the antibioticresistance protein comprises a tetracycline resistance protein or anoxytetracycline-resistance protein.
 44. A product of claim 42, whereinthe antibiotic resistance protein comprises a tetracycline resistanceprotein or an oxytetracycline-resistance protein.
 45. A product of claim43, wherein the antibiotic resistance protein is selected from the groupconsisting of OtrA, OtrB, OtrC, and Tcr3.
 46. A product of claim 44,wherein the antibiotic resistance protein is selected from the groupconsisting of OtrA, OtrB, OtrC, and Tcr3.
 47. A product of claim 31,which is V) the mutant host cell, which further comprises one or moresecond polynucleotides integrated into a chromosome of the host cell,wherein the second polynucleotide is selected from the group consistingof: a third polynucleotide encoding a third polypeptide comprising alysine decarboxylase polypeptide, a fragment thereof or a mutantthereof, and a fourth polynucleotide encoding a fourth polypeptidecomprising a lysine biosynthesis polypeptide, a fragment thereof or amutant thereof.
 48. A product of claim 31, which is V) the mutant hostcell, wherein the one or more polynucleotide sequences are integratedinto the chromosome of the host cell via a PCR-mediated gene replacementmethod.
 49. A product of claim 31, which is III) the first expressionplasmid vector, wherein the first polynucleotide is selected from thegroup consisting of tet, tetA, tetB, tetC, tetD, tetE, tetF, tetG, tetH,tetJ, tetK, tetL, tetM, tetO, tetP(A), tetP(B), tetQ, tetS, tetT, tetU,tetV, tetW, tetX, tetY, tetZ, tetA30, fragments thereof, and mutantsthereof.
 50. A product of claim 31, which is IV) the transformant,wherein the first polynucleotide is selected from the group consistingof tet, tetA, tetB, tetC, tetD, tetE, tetF, tetG, tetH, tetJ, tetK,tetL, tetM, tetO, tetP(A), tetP(B), tetQ, tetS, tetT, tetU, tetV, tetW,tetX, tetY, tetZ, tetA30, fragments thereof, and mutants thereof.
 51. Aproduct of claim 31, which is V) the mutant host cell, wherein the firstpolynucleotide is selected from the group consisting of tet, tetA, tetB,tetC, tetD, tetE, tetF, tetG, tetH, tetJ, tetK, tetL, tetM, tetO,tetP(A), tetP(B), tetQ, tetS, tetT, tetU, tetV, tetW, tetX, tetY, tetZ,tetA30, fragments thereof, and mutants thereof.
 52. A product of claim49, wherein the first polynucleotide is selected from the groupconsisting of SEQ ID NO: 1 (tetA), SEQ ID NO: 29 (tetA (nt 1-558)) andSEQ ID NO: 31 (tetA (nt 1-291)).
 53. A product of claim 50, wherein thefirst polynucleotide is selected from the group consisting of SEQ ID NO:1 (tetA), SEQ ID NO: 29 (tetA (nt 1-558)) and SEQ ID NO: 31 (tetA (nt1-291)).
 54. A product of claim 51, wherein the first polynucleotide isselected from the group consisting of SEQ ID NO: 1 (tetA), SEQ ID NO: 29(tetA (nt 1-558)) and SEQ ID NO: 31 (tetA (nt 1-291)).
 55. A product ofclaim 31, which is III) the first expression plasmid vector, wherein thefirst polypeptide is selected from the group consisting of TetA, TetB,TetC, TetD, TetE, TetF, TetG, TetH, TetJ, TetK, TetL, TetM, TetO,TetP(A), TetP(B), TetQ, TetS, TetT, TetU, TetV, TetW, TetX, TetY, TetZ,or Tet30, fragments thereof, and mutants thereof.
 56. A product of claim31, which is IV) the transformant, wherein the first polypeptide isselected from the group consisting of TetA, TetB, TetC, TetD, TetE,TetF, TetG, TetH, TetJ, TetK, TetL, TetM, TetO, TetP(A), TetP(B), TetQ,TetS, TetT, TetU, TetV, TetW, TetX, TetY, TetZ, or Tet30, fragmentsthereof, and mutants thereof.
 57. A product of claim 31, which is V) themutant host cell, wherein the first polypeptide is selected from thegroup consisting of TetA, TetB, TetC, TetD, TetE, TetF, TetG, TetH,TetJ, TetK, TetL, TetM, TetO, TetP(A), TetP(B), TetQ, TetS, TetT, TetU,TetV, TetW, TetX, TetY, TetZ, or Tet30, fragments thereof, and mutantsthereof.
 58. A product of claim 52, wherein the first polypeptide isselected from the group consisting of SEQ ID NO: 2 (TetA), SEQ ID NO: 30(TetA (aa1-185)), and SEQ ID NO: 32 (TetA (aa1-96)).
 59. A product ofclaim 53, wherein the first polypeptide is selected from the groupconsisting of SEQ ID NO: 2 (TetA), SEQ ID NO: 30 (TetA (aa1-185)), andSEQ ID NO: 32 (TetA (aa1-96)).
 60. A product of claim 54, wherein thefirst polypeptide is selected from the group consisting of SEQ ID NO: 2(TetA), SEQ ID NO: 30 (TetA (aa1-185)), and SEQ ID NO: 32 (TetA(aa1-96)).
 61. A product of claim 35, wherein the third polynucleotideis selected from the group consisting of SEQ ID NO: 41 (cadA), SEQ IDNO: 42 (ldcC), SEQ ID NO: 8 (ldc2), fragments thereof, and mutantsthereof, and the fourth polynucleotide is selected from the groupconsisting of sucA, ppc, aspC, lysC, asd, dapA, dapB, dapD, argD, dapE,dapF, lysA, ddh, pntAB, cyoABE, gadAB, ybjE, gdhA, gltA, sucC, gadC,acnB, pflB, thrA, aceA, aceB, gltB, aceE, sdhA, murE, speE, speG, puuA,puuP, ygjG, fragments thereof, and mutants thereof.
 62. A product ofclaim 36, wherein the third polynucleotide is selected from the groupconsisting of SEQ ID NO: 41 (cadA), SEQ ID NO: 42 (ldcC), SEQ ID NO: 8(ldc2), fragments thereof, and mutants thereof, and the fourthpolynucleotide is selected from the group consisting of sucA, ppc, aspC,lysC, asd, dapA, dapB, dapD, argD, dapE, dapF, lysA, ddh, pntAB, cyoABE,gadAB, ybjE, gdhA, gltA, sucC, gadC, acnB, pflB, thrA, aceA, aceB, gltB,aceE, sdhA, murE, speE, speG, puuA, puuP, ygjG, fragments thereof, andmutants thereof.
 63. A product of claim 47, wherein the thirdpolynucleotide is selected from the group consisting of SEQ ID NO: 41(cadA), SEQ ID NO: 42 (ldcC), SEQ ID NO: 8 (ldc2), fragments thereof,and mutants thereof, and the fourth polynucleotide is selected from thegroup consisting of sucA, ppc, aspC, lysC, asd, dapA, dapB, dapD, argD,dapE, dapF, lysA, ddh, pntAB, cyoABE, gadAB, ybjE, gdhA, gltA, sucC,gadC, acnB, pflB, thrA, aceA, aceB, gltB, aceE, sdhA, murE, speE, speG,puuA, puuP, ygjG, fragments thereof, and mutants thereof.
 64. A productof claim 61, wherein the mutant of SEQ ID NO: 8 (ldc2) may be selectedfrom the group consisting of SEQ ID NO: 10 (ldc2 co-1), SEQ ID NO: 34(ldc2 co-1 C332G), SEQ ID NO: 35 (ldc2 co-1 A785C), SEQ ID NO: 36 (ldc2co-1 A795C), SEQ ID NO: 37 (ldc2 co-1 C332G/A785C), SEQ ID NO: 38 (ldc2co-1 C332G/A795C), SEQ ID NO: 39 (ldc2 co-1 A785C/A795C), and SEQ ID NO:40 (ldc2 co-1 C332G/A785C/A795C).
 65. A product of claim 62, wherein themutant of SEQ ID NO: 8 (ldc2) may be selected from the group consistingof SEQ ID NO: 10 (ldc2 co-1), SEQ ID NO: 34 (ldc2 co-1 C332G), SEQ IDNO: 35 (ldc2 co-1 A785C), SEQ ID NO: 36 (ldc2 co-1 A795C), SEQ ID NO: 37(ldc2 co-1 C332G/A785C), SEQ ID NO: 38 (ldc2 co-1 C332G/A795C), SEQ IDNO: 39 (ldc2 co-1 A785C/A795C), and SEQ ID NO: 40 (ldc2 co-1C332G/A785C/A795C).
 66. A product of claim 63, wherein the mutant of SEQID NO: 8 (ldc2) may be selected from the group consisting of SEQ ID NO:10 (ldc2 co-1), SEQ ID NO: 34 (ldc2 co-1 C332G), SEQ ID NO: 35 (ldc2co-1 A785C), SEQ ID NO: 36 (ldc2 co-1 A795C), SEQ ID NO: 37 (ldc2 co-1C332G/A785C), SEQ ID NO: 38 (ldc2 co-1 C332G/A795C), SEQ ID NO: 39 (ldc2co-1 A785C/A795C), and SEQ ID NO: 40 (ldc2 co-1 C332G/A785C/A795C). 67.A product of claim 35, wherein the third polypeptide is selected fromthe group consisting of SEQ ID NO: 6 (CadA), SEQ ID NO: 7 (LdcC), SEQ IDNO: 9 (Ldc2), fragments thereof, and mutants thereof, and the fourthpolypeptide is selected from the group consisting of SucA, Ppc, AspC,LysC, Asd, DapA, DapB, DapD, ArgD, DapE, DapF, LysA, Ddh, PntAB, CyoABE,GadAB, YbjE, GdhA, GltA, SucC, GadC, AcnB, PflB, ThrA, AceA, AceB, GltB,AceE, SdhA, MurE, SpeE, SpeG, PuuA, PuuP, and YgjG, fragments thereof,and mutants thereof.
 68. A product of claim 36, wherein the thirdpolypeptide is selected from the group consisting of SEQ ID NO: 6(CadA), SEQ ID NO: 7 (LdcC), SEQ ID NO: 9 (Ldc2), fragments thereof, andmutants thereof, and the fourth polypeptide is selected from the groupconsisting of SucA, Ppc, AspC, LysC, Asd, DapA, DapB, DapD, ArgD, DapE,DapF, LysA, Ddh, PntAB, CyoABE, GadAB, YbjE, GdhA, GltA, SucC, GadC,AcnB, PflB, ThrA, AceA, AceB, GltB, AceE, SdhA, MurE, SpeE, SpeG, PuuA,PuuP, and YgjG, fragments thereof, and mutants thereof.
 69. A product ofclaim 47, wherein the third polypeptide is selected from the groupconsisting of SEQ ID NO: 6 (CadA), SEQ ID NO: 7 (LdcC), SEQ ID NO: 9(Ldc2), fragments thereof, and mutants thereof, and the fourthpolypeptide is selected from the group consisting of SucA, Ppc, AspC,LysC, Asd, DapA, DapB, DapD, ArgD, DapE, DapF, LysA, Ddh, PntAB, CyoABE,GadAB, YbjE, GdhA, GltA, SucC, GadC, AcnB, PflB, ThrA, AceA, AceB, GltB,AceE, SdhA, MurE, SpeE, SpeG, PuuA, PuuP, and YgjG, fragments thereof,and mutants thereof.
 70. A product of claim 67, wherein the mutant ofSEQ ID NO: 9 (Ldc2) is selected from the group consisting of SEQ ID NO:11 (Ldc2 S111C), SEQ ID NO: 16 (Ldc2 N262T), SEQ ID NO: 17 (Ldc2 K265N),SEQ ID NO: 18 (Ldc2 S111C/N262T), SEQ ID NO: 19 (Ldc2 S111C/K265N), SEQID NO: 20 (Ldc2 N262T/K265N), and SEQ ID NO: 21 (Ldc2S111C/N262T/K265N).
 71. A product of claim 68, wherein the mutant of SEQID NO: 9 (Ldc2) is selected from the group consisting of SEQ ID NO: 11(Ldc2 S111C), SEQ ID NO: 16 (Ldc2 N262T), SEQ ID NO: 17 (Ldc2 K265N),SEQ ID NO: 18 (Ldc2 S111C/N262T), SEQ ID NO: 19 (Ldc2 S111C/K265N), SEQID NO: 20 (Ldc2 N262T/K265N), and SEQ ID NO: 21 (Ldc2S111C/N262T/K265N).
 72. A product of claim 69, wherein the mutant of SEQID NO: 9 (Ldc2) is selected from the group consisting of SEQ ID NO: 11(Ldc2 S111C), SEQ ID NO: 16 (Ldc2 N262T), SEQ ID NO: 17 (Ldc2 K265N),SEQ ID NO: 18 (Ldc2 S111C/N262T), SEQ ID NO: 19 (Ldc2 S111C/K265N), SEQID NO: 20 (Ldc2 N262T/K265N), and SEQ ID NO: 21 (Ldc2S111C/N262T/K265N).
 73. A product of claim 35, wherein the fourthpolypeptide is selected from the group consisting of SEQ ID NO: 3(LysC), SEQ ID NO: 4 (DapA), SEQ ID NO: 5 (LysA), SEQ ID NO: 22 (Asd),SEQ ID NO: 23 (DapB), SEQ ID NO: 24 (DapD), SEQ ID NO: 25 (AspC), SEQ IDNO: 26 (LysC-1), SEQ ID NO: 27 (LysC-2), and SEQ ID NO: 28 (S-LysC). 74.A product of claim 36, wherein the fourth polypeptide is selected fromthe group consisting of SEQ ID NO: 3 (LysC), SEQ ID NO: 4 (DapA), SEQ IDNO: 5 (LysA), SEQ ID NO: 22 (Asd), SEQ ID NO: 23 (DapB), SEQ ID NO: 24(DapD), SEQ ID NO: 25 (AspC), SEQ ID NO: 26 (LysC-1), SEQ ID NO: 27(LysC-2), and SEQ ID NO: 28 (S-LysC).
 75. A product of claim 47, whereinthe fourth polypeptide is selected from the group consisting of SEQ IDNO: 3 (LysC), SEQ ID NO: 4 (DapA), SEQ ID NO: 5 (LysA), SEQ ID NO: 22(Asd), SEQ ID NO: 23 (DapB), SEQ ID NO: 24 (DapD), SEQ ID NO: 25 (AspC),SEQ ID NO: 26 (LysC-1), SEQ ID NO: 27 (LysC-2), and SEQ ID NO: 28(S-LysC).
 76. A product of claim 31, which is III) the first expressionplasmid vector, wherein the host cell is from the genus Hafnia,Escherichia, or Corynebacterium.
 77. A product of claim 31, which is IV)the transformant, wherein the host cell is from the genus Hafnia,Escherichia, or Corynebacterium.
 78. A product of claim 31, which is V)the mutant host cell, wherein the host cell is from the genus Hafnia,Escherichia, or Corynebacterium.
 79. A product of claim 76, wherein thehost cell is from the species Hafnia alvei, Escherichia coli, orCorynebacterium glutamicum.
 80. A product of claim 77, wherein the hostcell is from the species Hafnia alvei, Escherichia coli, orCorynebacterium glutamicum.
 81. A product of claim 78, wherein the hostcell is from the species Hafnia alvei, Escherichia coli, orCorynebacterium glutamicum.
 82. A method for producing a lysinecomprising: obtaining the transformant of the product IV) and/or themutant host cell of the product V) of claim 31; culturing thetransformant and/or mutant host cell under conditions effective for theexpression of the lysine; and harvesting the lysine.
 83. A method forproducing cadaverine (1,5-pentanediamine) comprising: 1a) cultivatingthe transformant of the product IV) and/or the mutant host cell of theproduct V) of claim 31; 1b) producing cadaverine using the cultureobtained from step 1a to decarboxylate lysine; and 1c) extracting andpurifying cadaverine using the culture obtained from step 1b.